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US20220340895A1 - Compositions and methods for treating leber's hereditary optic neuropathy - Google Patents

Compositions and methods for treating leber's hereditary optic neuropathy Download PDF

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US20220340895A1
US20220340895A1 US17/317,295 US202117317295A US2022340895A1 US 20220340895 A1 US20220340895 A1 US 20220340895A1 US 202117317295 A US202117317295 A US 202117317295A US 2022340895 A1 US2022340895 A1 US 2022340895A1
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pharmaceutical composition
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Bin Li
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Wuhan Neurophth Biotechnology Ltd Co
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Wuhan Neurophth Biotechnology Ltd Co
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Priority claimed from CN201810702492.7A external-priority patent/CN110656117A/en
Priority claimed from CN201810703168.7A external-priority patent/CN110724695A/en
Priority claimed from PCT/CN2018/095023 external-priority patent/WO2020010491A1/en
Priority claimed from CN201810948193.1A external-priority patent/CN110846392A/en
Priority claimed from PCT/CN2018/103937 external-priority patent/WO2020000641A1/en
Priority claimed from CN201811221305.XA external-priority patent/CN111073899B/en
Priority claimed from CN201811230856.2A external-priority patent/CN111068071A/en
Priority claimed from PCT/CN2019/094136 external-priority patent/WO2020001657A1/en
Priority to US17/317,295 priority Critical patent/US20220340895A1/en
Application filed by Wuhan Neurophth Biotechnology Ltd Co filed Critical Wuhan Neurophth Biotechnology Ltd Co
Assigned to Wuhan Neurophth Biotechnology Limited Company reassignment Wuhan Neurophth Biotechnology Limited Company CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: WUHAN NEUROPHTH BIOLOGICAL TECHNOLOGY LIMITED COMPANY
Assigned to WUHAN NEUROPHTH BIOLOGICAL TECHNOLOGY LIMITED COMPANY reassignment WUHAN NEUROPHTH BIOLOGICAL TECHNOLOGY LIMITED COMPANY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LI, BIN
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Definitions

  • LHON Lebers hereditary optic neuropathy
  • RRCs retinal ganglion cells
  • mtDNA pathogenic mitochondrial DNA
  • mutations are at nucleotide positions 11778 G to A (G11778A), 3460 G to A (G3460A) and 14484 T to C (TI 4484C), respectively in the NADH dehydrogenase subunit-4 protein (ND4), NADH dehydrogenase subunit-1 protein (ND1) and NADH dehydrogenase subunit-6 protein (ND6) subunit genes of complex I of the oxidative phosphorylation chain in mitochondria.
  • ND4 NADH dehydrogenase subunit-4 protein
  • ND1 NADH dehydrogenase subunit-1 protein
  • ND6 NADH dehydrogenase subunit-6 protein
  • Each mutation is believed to have significant risk of permanent loss of vision. It typically progresses within several weeks to several months without pain, until the binocular vision deteriorate to below 0.1, which seriously affects the quality of life of the patient.
  • Two LHON mutants results in the reduction of the patient's platelets isolated mitochondrial NADH dehydrogenase activity by 80%.
  • Ninety percent of the Chinese LHON patients carry the G11778A mutation.
  • the G11778A mutation changes an arginine into histidine in the ND4 protein, resulting the dysfunction and optic nerve damage in LHON patients.
  • a recombinant nucleic acid comprising: a mitochondrial targeting sequence; a mitochondrial protein coding sequence comprising a sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NO: 7, 8, 10, and 12; and a 3′UTR nucleic acid sequence.
  • the mitochondrial targeting sequence encodes a polypeptide comprising a peptide sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 129-159. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 2. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 3.
  • the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 4. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 5.
  • the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 7 or 8. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 10. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 12.
  • the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 111-125. In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14.
  • the recombinant nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 17-20, 23-24, 27-28, 31-34, 37-38, 41-42, 45-48, 51-52, 55-56, 59-62, 65-66, 69-70, 73-76, 79-80, and 83-84.
  • a recombinant nucleic acid comprising: a mitochondrial targeting sequence comprising a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, and 5; a mitochondrial protein coding sequence, wherein the mitochondrial protein coding sequence encodes a polypeptide comprising a mitochondrial protein; and a 3′UTR nucleic acid sequence.
  • the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 2. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 3. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 4. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 5.
  • the mitochondrial protein is selected from a group consisting of NADH dehydrogenase 4 (ND4), NADH dehydrogenase 6 (ND6), NADH dehydrogenase 1 (ND1), and a variant thereof.
  • the mitochondrial protein comprises NADH dehydrogenase 4 (ND4), or a variant thereof.
  • the mitochondrial protein comprises a peptide sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 160.
  • the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 6, 7, or 8.
  • the mitochondrial protein comprises NADH dehydrogenase 6 (ND6), or a variant thereof.
  • the mitochondrial protein comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 161.
  • the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 9 or 10.
  • the mitochondrial protein comprises NADH dehydrogenase 1 (ND1), or a variant thereof. In some cases, the mitochondrial protein comprises a sequence that is at least 90/o, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 162. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 11 or 12.
  • the 3′UTR nucleic acid sequence is located at 3′ of the mitochondrial targeting sequence.
  • the 3′UTR nucleic acid sequence comprises a sequence selected from the group consisting of hsACO2, hsATP5B, hsAK2, hsALDH2, hsCOX10, hsUQCRFS1, hsNDUFV1, hsNDUFV2, hsSOD2, hsCOX6c, hsIRPl, hsMRPS12, hsATP5J2, mSOD2, and hsOXA1L.
  • the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 111-125. In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14.
  • the mitochondrial targeting sequence is located at 5′ of the 3′UTR nucleic acid sequence. In some cases, the mitochondrial targeting sequence is located at 3′ of the mitochondrial targeting sequence.
  • the recombinant nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 29-84.
  • a recombinant nucleic acid comprising: a mitochondrial targeting sequence; a mitochondrial protein coding sequence comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 7, 8, 10, and 12; and a 3′UTR nucleic acid sequence.
  • the mitochondrial targeting sequence comprises a sequence encodes a polypeptide selected from the group consisting of hsCOX10, hsCOX8, scRPM2, lcSirt5, tbNDUS7, ncQCR2, hsATP5G2, hsLACTB, spilv1, gmCOX2, crATP6, hsOPA1, hsSDHD, hsADCK3, osP0644B06.24-2, Neurospora crassa ATP9 (ncATP9), hsGHITM, hsNDUFAB1, hsATP5G3, crATP6_hsADCK3, ncATP9_ncATP9, zmLOC100282174, ncATP9_zmLOC100282174_spilv1_ncATP9, zmLOC100282174_hsADCK3_crATP6_hsATP5G3, zmLOCIO0282174_h
  • the mitochondrial targeting sequence encodes a polypeptide comprising a peptide sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 129-159.
  • the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 2 or 3.
  • the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 4.
  • the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 5.
  • the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 7 or 8. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 10. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 12.
  • the 3′UTR nucleic acid sequence is located at 3′ of the mitochondrial targeting sequence.
  • the 3′UTR nucleic acid sequence comprises a sequence selected from the group consisting of hsACO2, hsATPSB, hsAK2, hsALDH2, hsCOX10, hsUQCRFS1, hsNDUFV1, hsNDUFV2, hsSOD2, hsCOX6c, hs1RP1, hsMRPS12, hsATP5J2, mSOD2, and hsOXA1L.
  • the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 111-125. In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14.
  • the mitochondrial targeting sequence is located at 5′ of the 3′UTR nucleic acid sequence. In some cases, the mitochondrial targeting sequence is located at 3′ of the mitochondrial targeting sequence.
  • the recombinant nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 17-20, 23-24, 27-28, 31-34, 37-38, 41-42, 4548, 51-52, 55-56, 59-62, 65-66, 69-70, 73-76, 79-80, and 83-84.
  • a recombinant nucleic acid comprising a mitochondrial targeting sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 2, 3, and 4.
  • the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 2.
  • the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 3.
  • the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 4.
  • the recombinant nucleic acid further comprises a mitochondrial protein coding sequence, wherein the mitochondrial protein coding sequence encodes a polypeptide comprising a mitochondrial protein.
  • the mitochondrial protein is selected from a group consisting of NADH dehydrogenase 4 (ND4), NADH dehydrogenase 6 (ND6), NADH dehydrogenase 1 (ND1), and a variant thereof.
  • the mitochondrial protein comprises NADH dehydrogenase 4 (ND4), or a variant thereof.
  • the mitochondrial protein comprises a peptide sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 160.
  • the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 6, 7, or 8.
  • the mitochondrial protein comprises NADH dehydrogenase 6 (ND6), or a variant thereof.
  • the mitochondrial protein comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 161.
  • the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 9 or 10.
  • the mitochondrial protein comprises NADH dehydrogenase 1 (ND1), or a variant thereof.
  • the mitochondrial protein comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 162.
  • the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 11 or 12.
  • the recombinant nucleic acid further comprises a 3′UTR nucleic acid sequence.
  • the 3′UTR nucleic acid sequence is located at 3′ of the mitochondrial targeting sequence.
  • the 3′UTR nucleic acid sequence comprises a sequence selected from the group consisting of hsACO2, hsATP5B, hsAK2, hsALDH2, hsCOX10, hsUQCRFS1, hsNDUFV1, hsNDUFV2, hsSOD2, hsCOX6c, hsIRPl, hsMRPS12, hsATP5J2, mSOD2, and hsOXA1L.
  • the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 111-125. In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14. In some cases, the mitochondrial targeting sequence is located at 5′ of the 3′UTR nucleic acid sequence. In some cases, the mitochondrial targeting sequence is located at 3′ of the mitochondrial targeting sequence.
  • the recombinant nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 29-70.
  • a recombinant nucleic acid comprising a mitochondrial protein coding sequence, wherein the mitochondrial protein coding sequence encodes a polypeptide comprising a mitochondrial protein, wherein the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 7, 8, 10, and 12.
  • the recombinant nucleic acid further comprises a mitochondrial targeting sequence.
  • the mitochondrial targeting sequence comprises a sequence encodes a polypeptide selected from the group consisting of hsCOX10, hsCOX8, scRPM2, lcSirt5, tbNDUS7, ncQCR2, hsATP5G2, hsLACTB, spilv1, gmCOX2, crATP6, hsOPA1, hsSDHD, hsADCK3, osP0644B06.24-2, Neurospora crassa ATP9 (ncATP9), hsGHITM, hsNDUFAB1, hsATP5G3, crATP6_hsADCK3, ncATP9_ncATP9, zmLOC100282174, ncATP9_zmLOC100282174_spily i_ncATP9, zmLOC100282174_hsADCK3
  • the mitochondrial targeting sequence encodes a polypeptide comprising a peptide sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 129-159. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 2. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 3.
  • the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 4. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 5.
  • the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 7 or 8. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 10. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97/a, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 12.
  • the recombinant nucleic acid further comprises a3′UTR nucleic acid sequence.
  • the 3′UTR nucleic acid sequence is located at 3′ of the mitochondrial targeting sequence.
  • the 3′UTR nucleic acid sequence comprises a sequence selected from the group consisting of hsACO2, hsATPSB, hsAK2, hsALDH2, hsCOX10, hsUQCRFS1, hsNDUFV1, hsNDUFV2, hsSOD2, hsCOX6c, hslRP1, hsMRPS12, hsATP5J2, mSOD2, and hsOXA1L.
  • the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 111-125. In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14. In some cases, the mitochondrial targeting sequence is located at 5′ of the 3′UTR nucleic acid sequence. In some cases, the mitochondrial targeting sequence is located at 3′ of the mitochondrial targeting sequence.
  • the recombinant nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 17-20, 23-24, 27-28, 31-34, 37-38, 41-42, 45-48, 51-52, 55-56, 59-62, 65-66, 69-70, 73-76, 79-80, and 83-84.
  • a viral vector comprising the recombinant nucleic acid disclosed herein.
  • the viral vector is an adeno-associated virus (AAV) vector.
  • AAV vector is selected from the group consisting ofAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16 vectors.
  • the AAV vector is a recombinant AAV (rAAV) vector.
  • the rAAV vector is rAAV2 vector.
  • a pharmaceutical composition comprising an adeno-associated virus (AAV) comprising any recombinant nucleic acid disclosed herein.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable excipient thereof.
  • a pharmaceutical composition comprising the viral vector disclosed herein, and a pharmaceutically acceptable excipient thereof, wherein the viral vector comprises any recombinant nucleic acid disclosed herein.
  • compositions comprising: an adeno-associated virus (AAV) comprising any recombinant nucleic acid disclosed herein, wherein the recombinant nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 15; and a pharmaceutically acceptable excipient.
  • AAV adeno-associated virus
  • the pharmaceutically acceptable excipient comprises phosphate-buffered saline (PBS), ⁇ , ⁇ -trehalose dehydrate, L-histidine monohydrochloride monohydrate, polysorbate 20, NaCl, NaH 2 PO 4 . Na 2 HPO 4 , KH 2 PO 4 , K 2 HPO 4 , poloxamer 188, or any combination thereof.
  • PBS phosphate-buffered saline
  • ⁇ , ⁇ -trehalose dehydrate ⁇ , ⁇ -trehalose dehydrate
  • L-histidine monohydrochloride monohydrate polysorbate 20
  • NaCl NaH 2 PO 4 .
  • Na 2 HPO 4 , KH 2 PO 4 , K 2 HPO 4 , poloxamer 188 poloxamer 188, or any combination thereof.
  • the pharmaceutically acceptable excipient is selected from phosphate-buffered saline (PBS), ⁇ , ⁇ -trehalose dehydrate, L-histidine monohydrochloride monohydrate, polysorbate 20, NaCl, NaH 2 PO 4 , Na 2 HPO 4 , KH 2 PO 4 , K 2 HPO 4 , poloxamer 188, and any combination thereof.
  • the pharmaceutically acceptable excipient comprises poloxamer 188.
  • the pharmaceutically acceptable excipient comprises 0.0001%-0.01% poloxamer 188.
  • the pharmaceutically acceptable excipient comprises 0.001% poloxamer 188.
  • the pharmaceutically acceptable excipient further comprises one or more salts.
  • the one or more salts comprises NaCl, NaH 2 PO 4 , Na 2 HPO 4 , and KH 2 PO 4 . In some cases, the one or more salts comprises 80 mM NaCl, 5 mM NaH 2 PO 4 , 40 mM Na 2 HPO 4 , and 5 mM KH 2 PO 4 . In some cases, the pharmaceutical composition has a pH of 6-8. In some cases, the pharmaceutical composition has a pH of 7.2-7.4. In some cases, the pharmaceutical composition has a pH of 7.3. In some cases, the pharmaceutical composition has a viral titer of at least 1.0 ⁇ 10 10 vg/mL. In some cases, the pharmaceutical composition has a viral titer of at least 5.0 ⁇ 10 10 vg/mL.
  • the pharmaceutical composition is subject to five freeze/thaw cycles, the pharmaceutical composition retains at least 60%, 70%, 80%, or 90% of a viral titer as compared to the viral titer prior to the five freeze/thaw cycles.
  • the pharmaceutical composition when administered to a patient with Leber's hereditary optic neuropathy, generates a higher average recovery of vision than a comparable pharmaceutical composition without the recombinant nucleic acid.
  • the pharmaceutical composition when administered to a patient with Leber's hereditary optic neuropathy, generates a higher average recovery of vision than a comparable pharmaceutical composition comprising a recombinant nucleic acid as set forth in SEQ ID NO: 15.
  • a method of treating an eye disorder comprising administering any pharmaceutical composition disclosed herein to a patient in need thereof.
  • the eye disorder is Leber's hereditary optic neuropathy (LHON).
  • the method comprises administering the pharmaceutical composition to one or both eyes of the patient.
  • the pharmaceutical composition is administered via intraocular or intravitreal injection.
  • the pharmaceutical composition is administered via intravitreal injection.
  • about 0.01-0.1 mL of the pharmaceutical composition is administered via intravitreal injection.
  • about 0.05 mL of the pharmaceutical composition is administered via intravitreal injection.
  • the method further comprises administering methylprednisolone to the patient.
  • the methylprednisolone is administered prior to the intravitreal injection of the pharmaceutical composition.
  • the methylprednisolone is administered orally
  • the methylprednisolone is administered daily for at least 1, 2, 3, 4, 5, 6, or 7 days prior to the intravitreal injection of the pharmaceutical composition.
  • the methylprednisolone is administered daily.
  • the a daily dosage of about 32 mg/60 kg methylprednisolone is administered.
  • the methylprednisolone is administered after the intravitreal injection of the pharmaceutical composition.
  • the method further comprises administering creatine phosphate sodium to the patient. In some cases, the creatine phosphate sodium is administered intravenously. In some cases, the methylprednisolone is administered intravenously or orally. In some cases, the method comprises administering methylprednisolone intravenously for at least one day, which is followed by administering methylprednisolone orally for at least a week. In some cases, the method comprises administering methylprednisolone intravenously for about 3 days, which is followed by administering methylprednisolone orally for at least about 6 weeks. In some cases, the methylprednisolone is administered intravenously at a daily dose of about 80 mg/60 kg.
  • the administering the pharmaceutical composition generates a higher average recovery of vision than a comparable pharmaceutical composition without the recombinant nucleic acid. In some cases, the administering the pharmaceutical composition generates a higher average recovery of vision than a comparable pharmaceutical composition comprising a recombinant nucleic acid as set forth in SEQ ID NO: 15.
  • FIG. 1 shows the PCR nucleic acid electrophoresis verification of ND4 (lane A) and optimized ND4 (lane B) gene cloning results.
  • FIG. 2 shows the relative expression level comparison using qPCR between the rAAV2-opt_ND4 (left black column) and rAAV2-ND4 (right black column).
  • ⁇ -actin is the internal reference gene (white column).
  • FIG. 3 shows the relative expression level comparison using immunoblotting between the rAAV2-opt_ND4 (left black column) and rAAV2-ND4 (right black column).
  • ⁇ -actin is the internal reference gene (white column).
  • FIG. 4 shows the fundus photographic results for rabbits injected with rAAV2-opt_ND4 (right) and rAAV2-ND4 (left), respectively.
  • FIG. 5 shows the fundus photographic results for a patient before (left) and after (right) the injection with rAAV2-optimized ND4.
  • FIG. 6 shows EGFP expression levels of rAAV2-ND4 (left) and rAAV2-opt_ND4* (right).
  • FIG. 7 shows the ND4 expression in 293T cells: rAAV2-ND4 (left) and rAAV2-opt_ND4* (right).
  • FIG. 8 shows the relative ND4 expression in 293T cells: rAAV2-ND4 (left) and rAAV2-opt_ND4* (right).
  • FIG. 9 shows the ND4 expression in rabbit optic nerve cells: rAAV2-ND4 (left) and rAAV2-opt_ND4* (right).
  • FIG. 10 shows the relative ND4 expression in rabbit optic nerve cells: rAAV2-ND4 (left) and rAAV2-opt_ND4* (right).
  • FIG. 11 shows the fundus photographic results for rAAV2-ND4 (left) and rAAV2-opt_ND4* (right).
  • FIG. 12 shows the microscope inspection (HE staining) results for rAAV2-ND4 (left) and rAAV2-opt_ND4* (right).
  • FIG. 13 shows the fundus photographic results for rabbits injected with rAAV2-ND6 (A), rAAV-GFP (B) and PBS, respectively.
  • FIG. 14 shows the fundus photographic results for rabbits injected with rAAV2-opt_ND6 (A), rAAV2-ND6 (B), rAAV-EGFP (C), respectively.
  • FIG. 15 shows the relative ND6 expression in rabbit optic nerve cells: rAAV2-opt_ND6 (A), rAAV2-ND6 (B), and rAAV-EGFP (C).
  • FIG. 16 shows the relative ND6 expression by western blot: rAAV2-opt_ND6 (A), rAAV2-ND6 (B), and rAAV-EGFP (C).
  • FIG. 17 shows the relative ND1 expression in rabbit optic nerve cells: rAAV2-opt_ND1 (A), rAAV2-ND1 (B), and rAAV-EGFP (C).
  • FIG. 18 shows the relative ND1 expression by western blot: rAAV2-opt_ND1 (A), rAAV2-ND1 (B), and rAAV-EGFP (C).
  • ranges include the range endpoints. Additionally, every subrange and value within the range is present as if explicitly written out.
  • the term “about” and its grammatical equivalents in relation to a reference numerical value and its grammatical equivalents as used herein can include a range of values plus or minus 10% from that value, such as a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.
  • the amount “about 10” includes amounts from 9 to 11.
  • subject refers to a mammal that has been or will be the object of treatment, observation or experiment.
  • mammal is intended to have its standard meaning, and encompasses humans, dogs, cats, sheep, and cows, for example.
  • the methods described herein can be useful in both human therapy and veterinary applications.
  • the subject is a human.
  • treating encompasses administration of at least one compound disclosed herein, or a pharmaceutically acceptable salt thereof, to a mammalian subject, particularly a human subject, in need of such an administration and includes (i) arresting the development of clinical symptoms of the disease, such as cancer, (ii) bringing about a regression in the clinical symptoms of the disease, such as cancer, and/or (iii) prophylactic treatment for preventing the onset of the disease, such as cancer.
  • terapéuticaally effective amount of a chemical entity described herein refers to an amount effective, when administered to a human or non-human subject, to provide a therapeutic benefit such as amelioration of symptoms, slowing of disease progression, or prevention of disease.
  • nucleic acid and “polynucleotide” can be used interchangeably.
  • Table 1 discloses all the nucleic acid and polypeptide sequences disclosed herein.
  • the first column shows the SEQ ID NO of each sequence.
  • the second column describes the nucleic acid or polypeptide construct.
  • the construct COX10-ND6-3′UTR is a nucleic acid combining the nucleic acid sequences of COX10 (SEQ ID NO: 1), ND6 (SEQ ID NO: 9), and 3′UTR (SEQ ID NO: 13) (from 5′ to 3′ without linker between the nucleic acid sequences.
  • AAV Adeno-Associated Virus
  • Adeno-associated virus is a small virus that infects humans and some other primate species.
  • the compositions disclosed herein comprises firstly an adeno-associated virus (AAV) genome or a derivative thereof.
  • An AAV genome is a polynucleotide sequence which encodes functions needed for production of an AAV viral particle. These functions include those operating in the replication and packaging cycle for AAV in a host cell, including encapsidation of the AAV genome into an AAV viral particle.
  • Naturally occurring AAV viruses are replication-deficient and rely on the provision of helper functions in trans for completion of a replication and packaging cycle. Accordingly, the AAV genome of the vector of the invention is typically replication-deficient.
  • the AAV genome can be in single-stranded form, either positive or negative-sense, or alternatively in double-stranded form.
  • the use of a double-stranded form allows bypass of the DNA replication step in the target cell and so can accelerate transgene expression.
  • the AAV genome may be from any naturally derived serotype or isolate or Glade of AAV.
  • the AAV genome may be the full genome of a naturally occurring AAV virus.
  • AAV viruses occurring in nature may be classified according to various biological systems.
  • AAV viruses are referred to in terms of their serotype.
  • a serotype corresponds to a variant subspecies of AAV which owing to its profile of expression of capsid surface antigens has a distinctive reactivity which can be used to distinguish it from other variant subspecies.
  • a virus having a particular AAV serotype does not efficiently cross-react with neutralising antibodies specific for any other AAV serotype.
  • AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, also recombinant serotypes, such as Rec2 and Rec3, recently identified from primate brain.
  • a preferred serotype of AAV for use in the invention is AAV2.
  • Other serotypes of particular interest for use in the invention include AAV4, AAV5 and AAV8 which efficiently transduce tissue in the eye, such as the retinal pigmented epithelium.
  • the serotype of AAV which is used can be an AAV serotype which is not AAV4. Reviews of AAV serotypes may be found in Choi et al (Curr Gene Ther. 2005; 5(3); 299-310) and Wu et al (Molecular Therapy. 2006; 14(3), 316-327).
  • sequences of AAV genomes or of elements of AAV genomes including ITR sequences, rep or cap genes for use in the invention may be derived from the following accession numbers for AAV whole genome sequences: Adeno-associated virus 1 NC_002077, AF063497; Adeno-associated virus 2 NC_001401; Adeno-associated virus 3 NC_001729; Adeno-associated virus 3B NC_001863; Adeno-associated virus 4 NC_001829; Adeno-associated virus 5 Y18065, AF085716; Adeno-associated virus 6 NC_001862; Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; Avian AAV strain DA-1 NC 006263, AY629583; Bovine AAV NC_005889, AY388617.
  • AAV viruses may also be referred to in terms of clades or clones. This refers to the phylogenetic relationship of naturally derived AAV viruses, and typically to a phylogenetic group of AAV viruses which can be traced back to a common ancestor, and includes all descendants thereof. Additionally, AAV viruses may be referred to in terms of a specific isolate, i.e. a genetic isolate of a specific AAV virus found in nature. The term genetic isolate describes a population of AAV viruses which has undergone limited genetic mixing with other naturally occurring AAV viruses, thereby defining a recognisably distinct population at a genetic level.
  • Examples of clades and isolates of AAV that may be used in the invention include: Clade A: AAV1 NC_002077, AF063497, AAV6 NC_001862, Hu. 48 AY530611, Hu 43 AY530606, Hu 44 AY530607, Hu 46 AY530609; Clade B: Hu. 19 AY530584, Hu.
  • AAV5 capsid has been shown to transduce primate cone photoreceptors efficiently as evidenced by the successful correction of an inherited color vision defect (Mancuso et al., Nature 2009, 461:784-7).
  • AAV serotypes determines the tissue specificity of infection (or tropism) of an AAV virus. Accordingly, preferred AAV serotypes for use in AAV viruses administered to patients in accordance with the invention are those which have natural tropism for or a high efficiency of infection of target cells within eye in LHON. Thus, AAV serotypes for use in AAV viruses administered to patients can be ones which infect cells of the neurosensory retina and retinal pigment epithelium.
  • the AAV genome of a naturally derived serotype or isolate or Glade of AAV comprises at least one inverted terminal repeat sequence (ITR).
  • ITR sequence acts in cis to provide a functional origin of replication, and allows for integration and excision of the vector from the genome of a cell.
  • one or more ITR sequences flank the polynucleotide sequence encoding ND4, ND6, or ND1 or a variant thereof.
  • Preferred ITR sequences are those of AAV2, and variants thereof.
  • the AAV genome typically also comprises packaging genes, such as rep and/or cap genes which encode packaging functions for an AAV viral particle.
  • the rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof.
  • the cap gene encodes one or more capsid proteins such as VP1, VP2 and VP3 or variants thereof. These proteins make up the capsid of an AAV viral particle. Capsid variants are discussed below.
  • a promoter will be operably linked to each of the packaging genes.
  • specific examples of such promoters include the p5, p19 and p40 promoters (Laughlin et al., 1979, PNAS, 76:5567-5571).
  • the p5 and p19 promoters are generally used to express the rep gene
  • the p40 promoter is generally used to express the cap gene.
  • the AAV genome used in the vector of the invention may therefore be the full genome of a naturally occurring AAV virus.
  • a vector comprising a full AAV genome may be used to prepare AAV virus in vitro.
  • the AAV genome will be derivatised for the purpose of administration to patients.
  • derivatisation is standard in the art and the present invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art. Derivatisation of the AAV genome and of the AAV capsid are reviewed in Coura and Nardi (Virology Journal, 2007, 4:99), and in Choi et al and Wu et al, referenced above.
  • Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a ND4, ND6, or ND1 transgene from a vector of the invention in vivo.
  • a derivative will include at least one inverted terminal repeat sequence (ITR), preferably more than one ITR, such as two ITRs or more.
  • ITRs may be derived from AAV genomes having different serotypes, or may be a chimeric or mutant ITR.
  • a preferred mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication of the genome to generate a single-stranded genome which contains both coding and complementary sequences i.e. a self-complementary AAV genome. This allows for bypass of DNA replication in the target cell, and so enables accelerated transgene expression.
  • the one or more ITRs will preferably flank the polynucleotide sequence encoding ND4, ND6, ND1, or a variant thereof at either end.
  • the inclusion of one or more ITRs is preferred to aid concatamer formation of the vector of the invention in the nucleus of a host cell, for example following the conversion of single-stranded vector DNA into double-stranded DNA by the action of host cell DNA polymerases.
  • the formation of such episomal concatamers protects the vector construct during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo.
  • ITR elements will be the only sequences retained from the native AAV genome in the derivative.
  • a derivative will preferably not include the rep and/or cap genes of the native genome and any other sequences of the native genome. This is preferred for the reasons described above, and also to reduce the possibility of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the transgene.
  • derivatives may additionally include one or more rep and/or cap genes or other viral sequences of an AAV genome.
  • Naturally occurring AAV virus integrates with a high frequency at a specific site on human chromosome 19, and shows a negligible frequency of random integration, such that retention of an integrative capacity in the vector may be tolerated in a therapeutic setting.
  • a derivative genome comprises genes encoding capsid proteins i.e. VP1.
  • VP2 and/or VP3 the derivative may be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAV viruses.
  • the invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones, or isolates of AAV within the same vector i.e. pseudotyping.
  • Chimeric, shuffled or capsid-modified derivatives will be typically selected to provide one or more desired functionalities for the viral vector.
  • these derivatives may display increased efficiency of gene delivery, decreased immunogenicity (humoral or cellular), an altered tropism range and/or improved targeting of a particular cell type compared to an AAV viral vector comprising a naturally occurring AAV genome, such as that of AAV2.
  • Increased efficiency of gene delivery may be effected by improved receptor or co-receptor binding at the cell surface, improved internalisation, improved trafficking within the cell and into the nucleus, improved uncoating of the viral particle and improved conversion of a single-stranded genome to double-stranded form.
  • Increased efficiency may also relate to an altered tropism range or targeting of a specific cell population, such that the vector dose is not diluted by administration to tissues where it is not needed.
  • Chimeric capsid proteins include those generated by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This may be performed for example by a marker rescue approach in which non-infectious capsid sequences of one serotype are cotransfected with capsid sequences of a different serotype, and directed selection is used to select for capsid sequences having desired properties.
  • the capsid sequences of the different serotypes can be altered by homologous recombination within the cell to produce novel chimeric capsid proteins.
  • Chimeric capsid proteins also include those generated by engineering of capsid protein sequences to transfer specific capsid protein domains, surface loops or specific amino acid residues between two or more capsid proteins, for example between two or more capsid proteins of different serotypes.
  • Hybrid AAV capsid genes can be created by randomly fragmenting the sequences of related AAV genes e.g. those encoding capsid proteins of multiple different serotypes and then subsequently reassembling the fragments in a self-priming polymerase reaction, which may also cause crossovers in regions of sequence homology.
  • a library of hybrid AAV genes created in this way by shuffling the capsid genes of several serotypes can be screened to identify viral clones having a desired functionality.
  • error prone PCR may be used to randomly mutate AAV capsid genes to create a diverse library of variants which may then be selected for a desired property.
  • capsid genes may also be genetically modified to introduce specific deletions, substitutions or insertions with respect to the native wild-type sequence.
  • capsid genes may be modified by the insertion of a sequence of an unrelated protein or peptide within an open reading frame of a capsid coding sequence, or at the N- and/or C-terminus of a capsid coding sequence.
  • the unrelated protein or peptide may advantageously be one which acts as a ligand for a particular cell type, thereby conferring improved binding to a target cell or improving the specificity of targeting of the vector to a particular cell population.
  • An example might include the use of RGD peptide to block uptake in the retinal pigment epithelium and thereby enhance transduction of surrounding retinal tissues (Cronin et al., 2008 ARVO Abstract: D1048).
  • the unrelated protein may also be one which assists purification of the viral particle as part of the production process i.e. an epitope or affinity tag.
  • the site of insertion will typically be selected so as not to interfere with other functions of the viral particle e.g. internalisation, trafficking of the viral particle. The skilled person can identify suitable sites for insertion based on their common general knowledge. Particular sites are disclosed in Choi et al, referenced above.
  • the invention additionally encompasses the provision of sequences of an AAV genome in a different order and configuration to that of a native AAV genome.
  • the invention also encompasses the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus.
  • Such chimeric genes may be composed of sequences from two or more related viral proteins of different viral species.
  • the vector of the invention takes the form of a polynucleotide sequence comprising an AAV genome or derivative thereof and a sequence encoding ND4, ND6, ND1 or a variant thereof.
  • the invention also provides an AAV viral particle comprising a vector of the invention.
  • the AAV particles of the invention include transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype.
  • the AAV particles of the invention also include mosaic forms wherein a mixture of unmodified capsid proteins from two or more different serotypes makes up the viral envelope.
  • the AAV particle also includes chemically modified forms bearing ligands adsorbed to the capsid surface.
  • such ligands may include antibodies for targeting a particular cell surface receptor.
  • the invention additionally provides a host cell comprising a vector or AAV viral particle of the invention.
  • nucleic acid sequences comprising a polynucleotide sequence encoding a NADH dehydrogenase subunit-4 (ND4), NADH dehydrogenase subunit-1 (ND1) and NADH dehydrogenase subunit-6 (ND6) polypeptide or a variant thereof.
  • ND4 NADH dehydrogenase subunit-4
  • ND1 NADH dehydrogenase subunit-1
  • ND6 NADH dehydrogenase subunit-6
  • the polynucleotide sequence for ND4 is shown in SEQ ID NO: 6 and encodes the protein shown in SEQ ID NO: 160. Further nucleic acid sequences for ND4 are SEQ ID NO: 7 and 8. The polynucleotide sequence for ND6 is shown in SEQ ID NO: 9 and encodes the protein shown in SEQ ID NO: 161. A further nucleic acid sequence for ND6 is SEQ ID NO: 10. The polynucleotide sequence for ND1 is shown in SEQ ID NO: 11 and encodes the protein shown in SEQ ID NO: 162. A further nucleic acid sequence for ND1 is SEQ ID NO: 12.
  • a variant of any one of SEQ ID NO: 160, 161, or 162 may comprise truncations, mutants or homologues thereof, and any transcript variants thereof which encode a functional ND4, ND6, or ND1 polypeptide.
  • Any homologues mentioned herein are typically at least 70% homologous to a relevant region of ND4, ND6, or ND1, and can functionally compensate for the polypeptide deficiency.
  • Homology can be measured using known methods.
  • the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et at (1984) Nucleic Acids Research 12, 387-395).
  • the PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et at (1990) J Mol Biol 215:403-10.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • a recombinant nucleic acid sequence may encode a polypeptide which is at least 55%, 65%, 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 97%, 99%, 99.5%, or 100% homologous to a relevant region of ND4, ND6, or ND1 (SEQ ID NO: 160, 161, or 162) over at least 20, preferably at least 30, for instance at least 40, 60, 100, 200, 300, 400 or more contiguous amino acids, or even over the entire sequence of the recombinant nucleic acid.
  • the relevant region will be one which provides for functional activity of ND4, ND6, or ND1.
  • the recombinant nucleic acid sequence may encode a polypeptide having at least 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 97%, 99%, 99.5%, or 100% homologous to full-length ND4, ND6, or ND1 (SEQ ID NO: 160, 161, or 162) over its entire sequence.
  • the recombinant nucleic acid sequence differs from the relevant region of ND4, ND6, or ND1 (SEQ ID NO: 160, 161, or 162) by at least, or less than, 2, 5, 10, 20, 40, 50 or 60 mutations (each of which can be substitutions, insertions or deletions).
  • a recombinant nucleic acid ND4, ND6, or ND1 polypeptide may have a percentage identity with a particular region of SEQ ID NO: 160, 161, or 162 which is the same as any of the specific percentage homology values (i.e. it may have at least 70%, 80% or 90% and more preferably at least 95%, 97/o, 99% identity) across any of the lengths of sequence mentioned above.
  • Variants of ND4, ND6, or ND1 also include truncations. Any truncation may be used so long as the variant is still functional. Truncations will typically be made to remove sequences that are non-essential for the protein activity and/or do not affect conformation of the folded protein, in particular folding of the active site. Appropriate truncations can routinely be identified by systematic truncation of sequences of varying length from the N- or C-terminus. Preferred truncations are N-terminal and may remove all other sequences except for the catalytic domain.
  • Variants of ND4, ND6, or ND1 further include mutants which have one or more, for example, 2, 3, 4, 5 to 10, 10 to 20, 20 to 40 or more, amino acid insertions, substitutions or deletions with respect to a particular region of ND4, ND6, or ND1 (SEQ ID NO: 160, 161, or 162).
  • Deletions and insertions are made preferably outside of the catalytic domain as described below. Substitutions are also typically made in regions that are non-essential for protease activity and/or do not affect conformation of the folded protein.
  • Substitutions preferably introduce one or more conservative changes, which replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume.
  • the amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace.
  • the conservative change may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid.
  • Conservative amino acid changes are well known in the art and may be selected in accordance with the properties of the amino acids.
  • ND6, or ND1 include polynucleotides having at least 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 96%, 97%, 98%, 99%, or 99.5% homologous to a relevant region of ND4, ND6, or ND1 (SEQ ID NO: 6, 9, or 11).
  • the variant displays these levels of homology to full-length ND4, ND6, or ND1 (SEQ ID NO: 6, 9, or 11) over its entire sequence.
  • Mitochondrial targeting sequences can be used to target proteins or mRNA to the mitochondria.
  • the charge, length, and structure of the MTS can be important for protein import into the mitochondria.
  • Particular 3′UTRs may drive mRNA localization to the mitochondrial surface and thus facilitate cotranslational protein import into the mitochondria.
  • the polynucleotide sequence for a mitochondrial targeting sequence can encode a polypeptide selected from hsCOX10, hsCOX8, scRPM2, lcSirt5, tbNDUS7, ncQCR2, hsATP5G2, hsLACTB, spilv1, gmCOX2, crATP6, hsOPA1, hsSDHD, hsADCK3, osP0644B06.24-2, Neurospora crassa ATP9 (ncATP9), hsGHITM, hsNDUFAB1, hsATP5G3, crATP6_hsADCK3, ncATP9_ncATP9, zmLOC100282174, ncATP9_zmLOC100282174_spilv1_ncATP9, zmLOCI00282174_hsADCK3_crATP6_hsATP5G3, zmLOC100282174_hs
  • the polynucleotide sequences, COX10 can encode the mitochondrial targeting sequence, MTS-COX10 (SEQ ID NO: 126).
  • the polynucleotide sequences, COX8 can encode the mitochondrial targeting sequence, MTS-COX8 (SEQ ID NO: 127).
  • the polynucleotide sequences, OPA1 can encode the mitochondrial targeting sequence, MTS-OPA 1 (SEQ ID NO: 128).
  • the 3′UTR nucleic acid sequence can be selected from hsACO2 (SEQ ID NO: 111), hsATP5B (SEQ ID NO: 112), hsAK2 (SEQ ID NO: 113), hsALDH2 (SEQ ID NO: 114), hsCOXI0 (SEQ ID NO: 115), hsUQCRFS1 (SEQ ID NO: 116), hsNDUFV1 (SEQ ID NO: 117), hsNDUFV2 (SEQ ID NO: 118), hsSOD2 (SEQ ID NO: 119), hsCOX6c (SEQ ID NO: 120), hsIRPl (SEQ ID NO: 121), hsMRPS12 (SEQ ID NO: 122), hsATP5J2 (SEQ ID NO: 123), mSOD2 (SEQ ID NO: 124), and hsOXA1L (SEQ ID NO: 125).
  • the 3′UTR nucleic acid sequence can also b e a variant having at least 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% homologous to any 3′UTR nucleic acid sequence listed here.
  • the 3′UTR nucleic acid sequence can be SEQ ID NO: 13 or 14.
  • recombinant nucleic acid sequences comprising a mitochondrial targeting sequence, a mitochondrial protein coding sequence, and a 3′UTR nucleic acid sequence.
  • the recombinant nucleic acid sequence can be selected from SEQ ID NO: 15-84.
  • the recombinant nucleic acid sequence can also be a variant having at least 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% homologous to any recombinant nucleic acid sequence listed here.
  • the vector of the invention also includes elements allowing for the expression of the disclosed transgene in vitro or in vivo.
  • the vector typically comprises a promoter sequence operably linked to the polynucleotide sequence encoding the ND4, ND6, or ND1 transgene or a variant thereof.
  • the promoter sequence may be constitutively active i.e. operational in any host cell background, or alternatively may be active only in a specific host cell environment, thus allowing for targeted expression of the transgene in a particular cell type.
  • the promoter may show inducible expression in response to presence of another factor, for example a factor present in a host cell. In any event, where the vector is administered for therapy, the promoter must be functional in a retinal cell background.
  • the promoter shows retinal-cell specific expression in order to allow for the transgene to only be expressed in retinal cell populations.
  • expression from the promoter may be retinal-cell specific, for example confined only to cells of the neurosensory retina and retinal pigment epithelium.
  • Preferred promoters for the ND4, ND6, or ND1 transgene include the chicken beta-actin (CBA) promoter, optionally in combination with a cytomegalovirus (CME) enhancer element.
  • CBA chicken beta-actin
  • CME cytomegalovirus
  • the preferred promoters for the ND4, ND6, or ND1 transgene comprises the CAG promoter.
  • a particularly preferred promoter is a hybrid CBA/CAG promoter, for example the promoter used in the rAVE expression cassette.
  • promoters based on human sequences that would induce retina specific gene expression include rhodospin kinase for rods and cones (Allocca et al., 2007, J Viol 81:11372-80), PR2.1 for cones only (Mancuso et al. 2009. Nature) and/or RPE65 for the retinal pigment epithelium (Bainbridge et al., 2008, N Eng J Med).
  • the vector of the invention may also comprise one or more additional regulatory sequences with may act pre- or post-transcriptionally.
  • the regulatory sequence may be part of the native ND4, ND6, or ND1 gene locus or may be a heterologous regulatory sequence.
  • the vector of the invention may comprise portions of the 5′UTR or 3′UTR from the native ND4, ND6, or ND1 transcript.
  • Regulatory sequences are any sequences which facilitate expression of the transgene i.e. act to increase expression of a transcript, improve nuclear export of mRNA or enhance its stability.
  • Such regulatory sequences include for example enhancer elements, postregulatory elements and polyadenylation sites.
  • a preferred polyadenylation site is the Bovine Growth Hormone poly-A signal.
  • the invention also encompasses the use of trans-acting regulatory sequences located on additional genetic constructs.
  • a preferred postregulatory element for use in a vector of the invention is the woodchuck hepatitis postregulatory element (WPRE) or a variant thereof.
  • WPRE woodchuck hepatitis postregulatory element
  • SAR scaffold-attachment region
  • the vector of the invention may be prepared by standard means known in the art for provision of vectors for gene therapy. Thus, well established public domain transfection, packaging and purification methods can be used to prepare a suitable vector preparation.
  • a vector of the invention may comprise the full genome of a naturally occurring AAV virus in addition to a polynucleotide sequence encoding ND4, ND6, or ND1 or a variant thereof.
  • a derivatised genome will be used, for instance a derivative which has at least one inverted terminal repeat sequence (ITR), but which may lack any AAV genes such as rcp or cap.
  • additional genetic constructs providing AAV and/or helper virus functions will be provided in a host cell in combination with the derivatised genome.
  • additional constructs will typically contain genes encoding structural AAV capsid proteins i.e. cap, VP1. VP2, VP3, and genes encoding other functions required for the AAV life cycle, such as rep.
  • the selection of structural capsid proteins provided on the additional construct will determine the serotype of the packaged viral vector.
  • a particularly preferred packaged viral vector for use in the invention comprises a derivatised genome of AAV2 in combination with AAV5 or AAV8 capsid proteins.
  • This packaged viral vector typically comprises one or more AAV2 ITRs.
  • AAV viruses are replication incompetent and so helper virus functions, preferably adenovirus helper functions will typically also be provided on one or more additional constructs to allow for AAV replication.
  • All of the above additional constructs may be provided as plasmids or other episomal elements in the host cell, or alternatively one or more constructs may be integrated into the genome of the host cell.
  • the invention provides a method for production of a vector of the invention.
  • the method comprises providing a vector which comprises an adeno-associated virus (AAV) genome or a derivative thereof and a polynucleotide sequence encoding ND4, ND6, or ND1 or a variant thereof in a host cell, and providing means for replication and assembly of the vector into an AAV viral particle.
  • the method comprises providing a vector comprising a derivative of an AAV genome and a polynucleotide sequence encoding ND4, ND6, or ND1 or a variant thereof, together with one or more additional genetic constructs encoding AAV and/or helper virus functions.
  • the derivative of an AAV genome comprises at least one ITR.
  • the method further comprises a step of purifying the assembled viral particles.
  • the method may comprise a step of formulating the viral particles for therapeutic use.
  • a vector of the invention may be used to address the cellular dysfunction underlying LHON.
  • they have shown that use of the vector can correct the defect associated with LHON. This provides a means whereby the degenerative process of the disease can be treated, arrested, palliated or prevented.
  • the invention therefore provides a method of treating or preventing LHON in a patient in need thereof, comprising administering a therapeutically effective amount of a vector of the invention to the patient by direct retinal, subretinal or intravitreal injection. Accordingly, LHON is thereby treated or prevented in the patient.
  • the invention provides for use of a vector of the invention in a method of treating or preventing LHON by administering said vector to a patient by direct retinal, subretinal or intravitreal injection. Additionally, the invention provides the use of a vector of the invention in the manufacture of a medicament for treating or preventing LHON by direct retinal, subretinal or intravitreal injection.
  • the vector of the invention may be administered in order to prevent the onset of one or more symptoms of LHON.
  • the patient may be asymptomatic.
  • the subject may have a predisposition to the disease.
  • the method or use may comprise a step of identifying whether or not a subject is at risk of developing, or has, LHON.
  • a prophylactically effective amount of the vector is administered to such a subject.
  • a prophylactically effective amount is an amount which prevents the onset of one or more symptoms of the disease.
  • the vector may be administered once the symptoms of the disease have appeared in a subject i.e. to cure existing symptoms of the disease.
  • a therapeutically effective amount of the antagonist is administered to such a subject.
  • a therapeutically effective amount is an amount which is effective to ameliorate one or more symptoms of the disease. Such an amount may also arrest, slow or reverse some loss of peripheral vision associated with LHON. Such an amount may also arrest, slow or reverse onset of LHON.
  • a typical single dose is between 10 10 and 10 12 genome particles, depending on the amount of remaining retinal tissue that requires transduction.
  • a genome particle is defined herein as an AAV capsid that contains a single stranded DNA molecule that can be quantified with a sequence specific method (such as real-time PCR). That dose may be provided as a single dose, but may be repeated for the fellow eye or in cases where vector may not have targeted the correct region of retina for whatever reason (such as surgical complication).
  • the treatment is preferably a single permanent treatment for each eye, but repeat injections, for example in future years and/or with different AAV serotypes may be considered.
  • the invention also provides a method of monitoring treatment or prevention of LHON in a patient comprising measuring activity ex vivo in retinal cells obtained from said patient following administration of the AAV vector of the invention by direct retinal, subretinal or intravitreal injection. This method can allow for determination of the efficacy of treatment.
  • the vector of the invention can be formulated into pharmaceutical compositions.
  • These compositions may comprise, in addition to the vector, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material may be determined by the skilled person according to the route of administration, i.e. here direct retinal, subretinal or intravitreal injection.
  • the pharmaceutical composition is typically in liquid form.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, a surfactant, such as pluronic acid (PF68) 0.001% may be used.
  • PF68 pluronic acid
  • the active ingredient will be in the form of an aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • the vector may be included in a pharmaceutical composition which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.
  • Samples that are suitable for use in the methods described herein can be nucleic acid samples from a subject.
  • a “nucleic acid sample” as used herein can include RNA or DNA, or a combination thereof.
  • a “polypeptide sample” e.g., peptides or proteins, or fragments therefrom
  • Nucleic acids and polypeptides can be extracted from one or more samples including but not limited to, blood, saliva, urine, mucosal scrapings of the lining of the mouth, expectorant, serum, tears, skin, tissue, or hair.
  • a nucleic acid sample can be assayed for nucleic acid information.
  • Nucleic acid information includes a nucleic acid sequence itself, the presence/absence of genetic variation in the nucleic acid sequence, a physical property which varies depending on the nucleic acid sequence (e.g., Tm), and the amount of the nucleic acid (e.g., number of mRNA copies).
  • a “nucleic acid” means any one of DNA, RNA, DNA including artificial nucleotides, or RNA including artificial nucleotides.
  • a “purified nucleic acid” includes cDNAs, fragments of genomic nucleic acids, nucleic acids produced using the polymerase chain reaction (PCR), nucleic acids formed by restriction enzyme treatment of genomic nucleic acids, recombinant nucleic acids, and chemically synthesized nucleic acid molecules.
  • a “recombinant” nucleic acid molecule includes a nucleic acid molecule made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
  • polypeptide includes proteins, fragments of proteins, and peptides, whether isolated from natural sources, produced by recombinant techniques, or chemically synthesized.
  • a polypeptide may have one or more modifications, such as a post-translational modification (e.g., glycosylation, phosphorylation, etc.) or any other modification (e.g., pegylation, etc.).
  • the polypeptide may contain one or more non-naturally-occurring amino acids (e.g., such as an amino acid with a side chain modification).
  • the nucleic acid sample can comprise cells or tissue, for example, cell lines.
  • Exemplary cell types from which nucleic acids can be obtained using the methods described herein include, but are not limited to, the following: a blood cell such as a B lymphocyte, T lymphocyte, leukocyte, erythrocyte, macrophage, or neutrophil; a muscle cell such as a skeletal cell, smooth muscle cell or cardiac muscle cell; a germ cell, such as a sperm or egg; an epithelial cell; a connective tissue cell, such as an adipocyte, chondrocyte; fibroblast or osteoblast; a neuron; an astrocyte; a stromal cell; an organ specific cell, such as a kidney cell, pancreatic cell, liver cell, or a keratinocyte; a stem cell; or any cell that develops therefrom.
  • a blood cell such as a B lymphocyte, T lymphocyte, leukocyte, erythrocyte, macrophage, or neutrophil
  • a cell from which nucleic acids can be obtained can be a blood cell or a particular type of blood cell including, for example, a hematopoietic stem cell or a cell that arises from a hematopoietic stem cell such as a red blood cell, B lymphocyte, T lymphocyte, natural killer cell, neutrophil, basophil, eosinophil, monocyte, macrophage, or platelet.
  • a hematopoietic stem cell such as a red blood cell, B lymphocyte, T lymphocyte, natural killer cell, neutrophil, basophil, eosinophil, monocyte, macrophage, or platelet.
  • stem cell can be used including, without limitation, an embryonic stem cell, adult stem cell, or pluripotent stem cell.
  • a nucleic acid sample can be processed for RNA or DNA isolation, for example, RNA or DNA in a cell or tissue sample can be separated from other components of the nucleic acid sample.
  • Cells can be harvested from a nucleic acid sample using standard techniques, for example, by centrifuging a cell sample and resuspending the pelleted cells, for example, in a buffered solution, for example, phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the cells after centrifuging the cell suspension to obtain a cell pellet, the cells can be lysed to extract DNA.
  • the nucleic acid sample can be concentrated and/or purified to isolate DNA.
  • nucleic acid samples obtained from a subject are considered to be obtained from the subject.
  • standard techniques and kits known in the art can be used to extract RNA or DNA from a nucleic acid sample, including, for example, phenol extraction, a QIAAMP® Tissue Kit (Qiagen, Chatsworth, Calif.), a WIZARD® Genomic DNA purification kit (Promega), or a Qiagen Autopure method using Puregene chemistry, which can enable purification of highly stable DNA well-suited for archiving.
  • determining the identity of an allele or determining copy number can, but need not, include obtaining a nucleic acid sample comprising RNA and/or DNA from a subject, and/or assessing the identity, copy number, presence or absence of one or more genetic variations and their chromosomal locations within the genomic DNA (i.e. subject's genome) derived from the nucleic acid sample.
  • the methods can include using information obtained by analysis of the nucleic acid sample by a third party.
  • the methods can include steps that occur at more than one site.
  • a nucleic acid sample can be obtained from a subject at a first site, such as at a health care provider or at the subject's home in the case of a self-testing kit.
  • the nucleic acid sample can be analyzed at the same or a second site, for example, at a laboratory or other testing facility.
  • nucleic acids and polypeptides described herein can be used in methods and kits of the present disclosure.
  • aptamers that specifically bind the nucleic acids and polypeptides described herein can be used in methods and kits of the present disclosure.
  • a nucleic acid can comprise a deoxyribonucleotide (DNA) or ribonucleotide (RNA), whether singular or in polymers, naturally occurring or non-naturally occurring, double-stranded or single-stranded, coding, for example a translated gene, or non-coding, for example a regulatory region, or any fragments, derivatives, mimetics or complements thereof.
  • nucleic acids can comprise oligonucleotides, nucleotides, polynucleotides, nucleic acid sequences, genomic sequences, complementary DNA (cDNA), antisense nucleic acids, DNA regions, probes, primers, genes, regulatory regions, introns, exons, open-reading frames, binding sites, target nucleic acids and allele-specific nucleic acids.
  • cDNA complementary DNA
  • a “probe,” as used herein, includes a nucleic acid fragment for examining a nucleic acid in a specimen using the hybridization reaction based on the complementarity of nucleic acid.
  • hybrid includes a double strand formed between any one of the abovementioned nucleic acid, within the same type, or across different types, including DNA-DNA, DNA-RNA, RNA-RNA or the like.
  • isolated nucleic acids are separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library).
  • isolated nucleic acids of the disclosure can be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the isolated material can form part of a composition, for example, a crude extract containing other substances, buffer system or reagent mix.
  • the material can be purified to essential homogeneity using methods known in the art, for example, by polyacrylamide gel electrophoresis (PAGE) or column chromatography (e.g., HPLC).
  • PAGE polyacrylamide gel electrophoresis
  • HPLC column chromatography
  • isolated also can refer to nucleic acids that are separated from the chromosome with which the genomic DNA is naturally associated.
  • the isolated nucleic acid molecule can contain less than about 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotides that flank the nucleic acid molecule in the gDNA of the cell from which the nucleic acid molecule is derived.
  • Nucleic acids can be fused to other coding or regulatory sequences can be considered isolated.
  • recombinant DNA contained in a vector is included in the definition of “isolated” as used herein.
  • isolated nucleic acids can include recombinant DNA molecules in heterologous host cells or heterologous organisms, as well as partially or substantially purified DNA molecules in solution. Isolated nucleic acids also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present disclosure.
  • An isolated nucleic acid molecule or nucleotide sequence can be synthesized chemically or by recombinant means.
  • nucleotide sequences can be useful, for example, in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene, in tissue (e.g., human tissue), such as by Northern blot analysis or other hybridization techniques disclosed herein.
  • tissue e.g., human tissue
  • the disclosure also pertains to nucleic acid sequences that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein
  • Such nucleic acid sequences can be detected and/or isolated by allele- or sequence-specific hybridization (e.g., under high stringency conditions).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, of the length of the reference sequence.
  • the actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm.
  • a non-limiting example of such a mathematical algorithm is described in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90-5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0), as described in Altschul, S. et al., Nucleic Acids Res., 25:3389-3402 (1997).
  • any relevant parameters of the respective programs can be used.
  • Other examples include the algorithm of Myers and Miller, CABIOS (1989). ADVANCE, ADAM, BLAT, and FASTA.
  • the percent identity between two amino acid sequences can be accomplished using, for example, the GAP program in the GCG software package (Accelrys, Cambridge, UK).
  • Probes can be oligonucleotides that hybridize in a base-specific manner to a complementary strand of a nucleic acid molecule.
  • Probes can include primers, which can be a single-stranded oligonucleotide probe that can act as a point of initiation of template-directed DNA synthesis using methods including but not limited to, polymerase chain reaction (PCR) and ligase chain reaction (LCR) for amplification of a target sequence.
  • Oligonucleotides, as described herein, can include segments or fragments of nucleic acid sequences, or their complements.
  • DNA segments can be between 5 and 10,000 contiguous bases, and can range from 5, 10, 12, 15, 20, or 25 nucleotides to 10, 15, 20, 25, 30, 40, 50, 100, 200, 500, 1000 or 10,000 nucleotides.
  • probes and primers can include polypeptide nucleic acids (PNA), as described in Nielsen, P. et al., Science 254: 1497-1500 (1991).
  • PNA polypeptide nucleic acids
  • a probe or primer can comprise a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and in certain embodiments about 40, 50, 60 or 75, consecutive nucleotides of a nucleic acid molecule.
  • the present disclosure also provides isolated nucleic acids, for example, probes or primers, that contain a fragment or portion that can selectively hybridize to a nucleic acid that comprises, or consists of, a nucleotide sequence, wherein the nucleotide sequence can comprise at least one polymorphism or polymorphic allele contained in the genetic variations described herein or the wild-type nucleotide that is located at the same position, or the complements thereof.
  • the probe or primer can be at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical, to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence.
  • a nucleic acid probe can be an oligonucleotide capable of hybridizing with a complementary region of a gene associated with a condition (e.g., LHON) containing a genetic variation described herein.
  • the nucleic acid fragments of the disclosure can be used as probes or primers in assays such as those described herein.
  • DNA can be amplified and/or can be labeled (e.g., radiolabeled, fluorescently labeled) and used as a probe for screening, for example, a cDNA library derived from an organism.
  • cDNA can be derived from mRNA and can be contained in a suitable vector.
  • corresponding clones can be isolated, DNA obtained fallowing in vivo excision, and the cloned insert can be sequenced in either or both orientations by art-recognized methods to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight.
  • the polypeptide and the DNA encoding the polypeptide can be isolated, sequenced and further characterized.
  • nucleic acid can comprise one or more polymorphisms, variations, or mutations, for example, single nucleotide polymorphisms (SNPs), single nucleotide variations (SNVs), copy number variations (CNVs), for example, insertions, deletions, inversions, and translocations.
  • SNPs single nucleotide polymorphisms
  • SNVs single nucleotide variations
  • CNVs copy number variations
  • nucleic acids can comprise analogs, for example, phosphorothioates, phosphoramidates, methyl phosphonate, chiralmethyl phosphonates, 2-O-methyl ribonucleotides, or modified nucleic acids, for example, modified backbone residues or linkages, or nucleic acids combined with carbohydrates, lipids, polypeptide or other materials, or peptide nucleic acids (PNAs), for example, chromatin, ribosomes, and transcriptosomes.
  • nucleic acids can comprise nucleic acids in various structures, for example, A DNA, B DNA, Z-form DNA, siRNA, tRNA, and ribozymes.
  • the nucleic acid may be naturally or non-naturally polymorphic, for example, having one or more sequence differences, for example, additions, deletions and/or substitutions, as compared to a reference sequence.
  • a reference sequence can be based on publicly available information, for example, the U.C. Santa Cruz Human Genome Browser Gateway (genome.ucsc.edu/cgi-bin/hgGateway) or the NCBI website (www.ncbi.nlm.nih.gov).
  • a reference sequence can be determined by a practitioner of the present disclosure using methods well known in the art, for example, by sequencing a reference nucleic acid.
  • a probe can hybridize to an allele, SNP, SNV, or CNV as described herein. In some embodiments, the probe can bind to another marker sequence associated with LHON as described herein.
  • Control probes can also be used, for example, a probe that binds a less variable sequence, for example, a repetitive DNA associated with a centromere of a chromosome, can be used as a control.
  • probes can be obtained from commercial sources.
  • probes can be synthesized, for example, chemically or in vitro, or made from chromosomal or genomic DNA through standard techniques.
  • sources of DNA that can be used include genomic DNA, cloned DNA sequences, somatic cell hybrids that contain one, or a part of one, human chromosome along with the normal chromosome complement of the host, and chromosomes purified by flow cytometry or microdissection.
  • the region of interest can be isolated through cloning, or by site-specific amplification using PCR.
  • a detectable label can comprise any label capable of detection by a physical, chemical, or a biological process for example, a radioactive label, such as 32P or 3H, a fluorescent label, such as FITC, a chromophore label, an afinity-ligand label, an enzyme label, such as alkaline phosphatase, horseradish peroxidase, or 12 galactosidase, an enzyme cofactor label, a hapten conjugate label, such as digoxigenin or dinitrophenyl, a Raman signal generating label, a magnetic label, a spin label, an epitope label, such as the FLAG or HA epitope, a luminescent label, a heavy atom label, a nanoparticle label, an electrochemical label, a light scattering label, a spherical shell label, semiconductor nanoc
  • a nucleotide can be directly incorporated into a probe with standard techniques, for example, nick translation, random priming, and PCR labeling.
  • a “signal,” as used herein, include a signal suitably detectable and measurable by appropriate means, including fluorescence, radioactivity, chemiluminescence, and the like.
  • Non-limiting examples of label moieties useful for detection include, without limitation, suitable enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; members of a binding pair that are capable of forming complexes such as streptavidin/biotin, avidin/biotin or an antigen/antibody complex including, for example, rabbit IgG and anti-rabbit IgG; fluorophores such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, tetramethyl rhodamine, cosin, green fluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue, Texas Red, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, fluorescent lanthanide complexes such
  • Lakowicz Editor
  • Plenum Pub Corp 2nd edition (July 1999) and the 6th Edition of the Molecular Probes Handbook by Richard P. Hoagland
  • a luminescent material such as luminol
  • light scattering or plasmon resonant materials such as gold or silver particles or quantum dots
  • radioactive material include 14C, 123I, 124I, 125I, Tc99m, 32P, 33P, 35S or 3H.
  • Backbone labels comprise nucleic acid stains that bind nucleic acids in a sequence independent manner.
  • Non-limiting examples include intercalating dyes such as phenanthridines and acridines (e.g., ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidium monoazide, and ACMA); some minor grove binders such as indoles and imidazoles (e.g., Hoechst 33258, Hoechst 33342, Hoechst 34580 and DAPI); and miscellaneous nucleic acid stains such as acridine orange (also capable of intercalating), 7-AAD, actinomycin D, LDS751, and hydroxystilbamidine.
  • intercalating dyes such as phenanthridines and acridines (e.g., ethidium bromide,
  • nucleic acid stains are commercially available from suppliers such as Molecular Probes, Inc. Still other examples of nucleic acid stains include the following dyes from Molecular Probes: cyanine dyes such as SYTOX Blue, SYTOX Green, SYTOX Orange.
  • fluorophores of different colors can be chosen, for example, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), 5-(and-6)-carboxy-X-rhodamine, lissamine rhodamine B, 5-(and-6)-carboxyfluorescein, fluoreseein-5-isothiocyanate (FITC), 7-diethylaminocoumarin-3-carboxvlic acid, tetramethvlrhodamine-5-(and-6)-isothiocvanate, 5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylic acid, 6-[fluorescein 5-(and-6)-carboxamido]hexanoic acid, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionic acid, cosin-5-isothiocyanate,
  • AMCA
  • fluorescently labeled probes can be viewed with a fluorescence microscope and an appropriate filter for each fluorophore, or by using dual or triple band-pass filter sets to observe multiple fluorophores.
  • techniques such as flow cytometry can be used to examine the hybridization pattern of the probes.
  • the probes can be indirectly labeled, for example, with biotin or digoxygenin, or labeled with radioactive isotopes such as 32P and/or 3H.
  • a probe indirectly labeled with biotin can be detected by avidin conjugated to a detectable marker.
  • avidin can be conjugated to an enzymatic marker such as alkaline phosphatase or horseradish peroxidase.
  • enzymatic markers can be detected using colorimetric rcactions using a substrate and/or a catalyst for the enzyme.
  • catalysts for alkaline phosphatase can be used, for example, 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.
  • a catalyst can be used for horseradish peroxidase, for example, diaminobenzoate.
  • compositions comprising an agent or combination of agents of the instant disclosure.
  • Such pharmaceutical compositions can be used to treat a condition (e.g., LHON) as described above.
  • Compounds of the disclosure can be administered as pharmaceutical formulations including those suitable for oral (including buccal and sub-lingual), rectal, nasal, topical, transdermal patch, pulmonary, vaginal, suppository, or parenteral (including intraocular, intravitreal, intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous and intravenous) administration or in a form suitable for administration by aerosolization, inhalation or insufflation.
  • parenteral including intraocular, intravitreal, intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous and intravenous
  • aerosolization inhalation or insufflation.
  • General information on drug delivery systems can be found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore Md. (1999).
  • the pharmaceutical composition includes carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, polypeptides, amino acids, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents and/or preservatives), water, oils including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline solutions, aqueous dextrose and glycerol solutions, flavoring agents, coloring agents, detackifiers and other acceptable additives, adjuvants, or binders, other pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH buffering agents, tonicity adjusting agents, emulsifying agents, wetting agents and the like.
  • carriers and excipients including but not limited to buffers, carbohydrates, mannitol, polypeptides, amino acids, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents and/or preservatives
  • water oils
  • excipients examples include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the pharmaceutical preparation is substantially free of preservatives.
  • the pharmaceutical preparation can contain at least one preservative.
  • General methodology on pharmaceutical dosage forms is found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott, Williams, & Wilkins, Baltimore Md. (1999)). It can be recognized that, while any suitable carrier known to those of ordinary skill in the art can be employed to administer the compositions of this disclosure, the type of carrier can vary depending on the mode of administration.
  • Biodegradable microspheres can also be employed as carriers for the pharmaceutical compositions of this disclosure. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268, 5,075,109, 5,928,647, 5,811,128, 5,820,883, 5,853,763, 5,814,344 and 5,942,252.
  • the compound can be administered in liposomes or microspheres (or microparticles).
  • Methods for preparing liposomes and microspheres for administration to a subject are well known to those of skill in the art.
  • U.S. Pat. No. 4,789,734 the contents of which are hereby incorporated by reference, describes methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, and along with surfactants if required, and the material dialyzed or sonicated, as necessary.
  • a review of known methods is provided by G. Gregoriadis, Chapter 14, “Liposomes.” Drug Carriers in Biology and Medicine, pp. 2.sup.87-341 (Academic Press, 1979).
  • Microspheres formed of polymers or polypeptides are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the blood stream. Alternatively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time ranging from days to months. See, for example, U.S. Pat. Nos. 4,906,474, 4,925,673 and 3,625,214, and Jein, TIPS 19:155-157 (1998), the contents of which are hereby incorporated by reference.
  • the concentration of drug can be adjusted, the pH of the solution buffered and the isotonicity adjusted to be compatible with intraocular or intravitreal injection.
  • the compounds of the disclosure can be formulated as a sterile solution or suspension, in suitable vehicles.
  • the pharmaceutical compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterile filtered.
  • the resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • Suitable formulations and additional carriers are described in Remington “The Science and Practice of Pharmacy” (20th Ed., Lippincott Williams & Wilkins, Baltimore Md.), the teachings of which are incorporated by reference in their entirety herein.
  • agents or their pharmaceutically acceptable salts can be provided alone or in combination with one or more other agents or with one or more other forms.
  • a formulation can comprise one or more agents in particular proportions, depending on the relative potencies of each agent and the intended indication. For example, in compositions for targeting two different host targets, and where potencies are similar, about a 1:1 ratio of agents can be used.
  • the two forms can be formulated together, in the same dosage unit e.g., in one cream, suppository, tablet, capsule, aerosol spray, or packet of powder to be dissolved in a beverage; or each form can be formulated in a separate unit, e.g., two creams, two suppositories, two tablets, two capsules, a tablet and a liquid for dissolving the tablet, two aerosol sprays, or a packet of powder and a liquid for dissolving the powder, etc.
  • pharmaceutically acceptable salt means those salts which retain the biological effectiveness and properties of the agents used in the present disclosure, and which are not biologically or otherwise undesirable.
  • Typical salts are those of the inorganic ions, such as, for example, sodium, potassium, calcium, magnesium ions, and the like.
  • Such salts include salts with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid or maleic acid.
  • the agent(s) if the agent(s) contain a carboxyl group or other acidic group, it can be converted into a pharmaceutically acceptable addition salt with inorganic or organic bases.
  • suitable bases include sodium hydroxide, potassium hydroxide, ammonia, cyclohexylamine, dicyclohexyl-amine, ethanolamine, diethanolamine, triethanolamine, and the like.
  • a pharmaceutically acceptable ester or amide refers to those which retain biological effectiveness and properties of the agents used in the present disclosure, and which are not biologically or otherwise undesirable.
  • Typical esters include ethyl, methyl, isobutyl, ethylene glycol, and the like.
  • Typical amides include unsubstituted amides, alkyl amides, dialkyl amides, and the like.
  • an agent can be administered in combination with one or more other compounds, forms, and/or agents. e.g., as described above.
  • Pharmaceutical compositions with one or more other active agents can be formulated to comprise certain molar ratios. For example, molar ratios of about 99:1 to about 1:99 of a first active agent to the other active agent can be used.
  • the range of molar ratios of a first active agent: other active agents are selected from about 80:20 to about 20:80; about 75:25 to about 25:75, about 70:30 to about 30:70, about 66:33 to about 33:66, about 60:40 to about 40:60; about 50:50; and about 90:10 to about 10:90.
  • the molar ratio of a first active: other active agents can be about 1:9, and in some embodiments can be about 1:1.
  • the two agents, forms and/or compounds can be formulated together, in the same dosage unit e.g., in one cream, suppository, tablet, capsule, or packet of powder to be dissolved in a beverage; or each agent, form, and/or compound can be formulated in separate units, e.g., two creams, suppositories, tablets, two capsules, a tablet and a liquid for dissolving the tablet, an aerosol spray a packet of powder and a liquid for dissolving the powder, etc.
  • agents and/or combinations of agents can be administered with still other agents.
  • the choice of agents that can be co-administered with the agents and/or combinations of agents of the instant disclosure can depend, at least in part, on the condition being treated.
  • the agent(s) can be administered per se or in the form of a pharmaceutical composition wherein the active agent(s) is in an admixture or mixture with one or more pharmaceutically acceptable carriers.
  • a pharmaceutical composition can be any composition prepared for administration to a subject.
  • Pharmaceutical compositions for use in accordance with the present disclosure can be formulated in conventional manner using one or more physiologically acceptable carriers, comprising excipients, diluents, and/or auxiliaries, e.g., which facilitate processing of the active agents into preparations that can be administered. Proper formulation can depend at least in part upon the route of administration chosen.
  • agent(s) useful in the present disclosure can be delivered to a subject using a number of routes or modes of administration, including oral, buccal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous, intraocular, intravitreal, and intramuscular applications, as well as by inhalation.
  • oils or non-aqueous solvents can be used to bring the agents into solution, due to, for example, the presence of large lipophilic moieties.
  • emulsions, suspensions, or other preparations for example, liposomal preparations.
  • liposomal preparations any known methods for preparing liposomes for treatment of a condition can be used. See, for example, Bangham et al., J. Mol. Biol. 23: 238-252 (1965) and Szoka et al., Proc. Natl Acad. Sci. USA 75: 4194-4198 (1978), incorporated herein by reference.
  • Ligands can also be attached to the liposomes to direct these compositions to particular sites of action.
  • Agents of this disclosure can also be integrated into foodstuffs, e.g., cream cheese, butter, salad dressing, or ice cream to facilitate solubilization, administration, and/or compliance in certain subject populations.
  • the compounds of the disclosure can be formulated for parenteral administration (e.g., by injection, for example, intraocular or intravitreal injection) and can be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative.
  • the compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example, solutions in aqueous polyethylene glycol.
  • the vehicle can be chosen from those known in art to be suitable, including aqueous solutions or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles.
  • the formulation can also comprise polymer compositions which are biocompatible, biodegradable, such as poly(lactic-co-glycolic)acid. These materials can be made into micro or nanospheres, loaded with drug and further coated or derivatized to provide superior sustained release performance.
  • Vehicles suitable for periocular or intraocular injection include, for example, suspensions of therapeutic agent in injection grade water, liposomes and vehicles suitable for lipophilic substances. Other vehicles for periocular or intraocular injection are well known in the art.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
  • the active compound When administration is by injection, the active compound can be formulated in aqueous solutions, specifically in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer.
  • the solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active compound can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the pharmaceutical composition does not comprise an adjuvant or any other substance added to enhance the immune response stimulated by the peptide.
  • the pharmaceutical composition comprises a substance that inhibits an immune response to the peptide. Methods of formulation are known in the art, for example, as disclosed in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton P.
  • eye disorders can be effectively treated with ophthalmic solutions, suspensions, ointments or inserts comprising an agent or combination of agents of the present disclosure.
  • Eye drops can be prepared by dissolving the active ingredient in a sterile aqueous solution such as physiological saline, buffering solution, etc., or by combining powder compositions to be dissolved before use.
  • Other vehicles can be chosen, as is known in the art, including but not limited to: balance salt solution, saline solution, water soluble polyethers such as polyethyene glycol, polyvinyls, such as polyvinyl alcohol and povidone, cellulose derivatives such as methylcellulose and hydroxypropyl methylcellulose, petroleum derivatives such as mineral oil and white petrolatum, animal fats such as lanolin, polymers of acrylic acid such as carboxypolymethylene gel, vegetable fats such as peanut oil and polysaccharides such as dextrans, and glycosaminoglycans such as sodium hyaluronate. If desired, additives ordinarily used in the eye drops can be added.
  • water soluble polyethers such as polyethyene glycol
  • polyvinyls such as polyvinyl alcohol and povidone
  • cellulose derivatives such as methylcellulose and hydroxypropyl methylcellulose
  • petroleum derivatives such as mineral oil and white petrolatum
  • animal fats such as
  • Such additives include isotonizing agents (e.g., sodium chloride, etc.), buffer agent (e.g., boric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc.), preservatives (e.g., benzalkonium chloride, benzethonium chloride, chlorobutanol, etc.), thickeners (e.g., saccharide such as lactose, mannitol, maltose, etc.; e.g., hyaluronic acid or its salt such as sodium hyaluronate, potassium hyaluronate, etc.; e.g., mucopolysaccharide such as chondroitin sulfate, etc.; e.g., sodium polyacrylate, carboxyvinyl polymer, crosslinked polyacrylate, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl methylcellulose, hydroxyethyl
  • solubility of the components of the present compositions can be enhanced by a surfactant or other appropriate co-solvent in the composition.
  • cosolvents include polysorbate 20, 60, and 80, Pluronic F68, F-84 and P-103, cyclodextrin, or other agents known to those skilled in the art.
  • co-solvents can be employed at a level of from about 0.01% to 2% by weight.
  • compositions of the disclosure can be packaged in multidose form.
  • Preservatives can be preferred to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, Onamer M. or other agents known to those skilled in the art. In the prior art ophthalmic products, such preservatives can be employed at a level of from 0.004% to 0.02%.
  • the preservative preferably benzalkonium chloride
  • the preservative can be employed at a level of from 0.001% to less than 0.01%, e.g., from 0.001% to 0.008%, preferably about 0.005% by weight. It has been found that a concentration of benzalkonium chloride of 0.005% can be sufficient to preserve the compositions of the present disclosure from microbial attack.
  • the agents of the present disclosure are delivered in soluble rather than suspension form, which allows for more rapid and quantitative absorption to the sites of action.
  • formulations such as jellies, creams, lotions, suppositories and ointments can provide an area with more extended exposure to the agents of the present disclosure, while formulations in solution, e.g., sprays, provide more immediate, short-term exposure.
  • the compounds of the disclosure can be attached releasably to biocompatible polymers for use in sustained release formulations on, in or attached to inserts for topical, intraocular, periocular, or systemic administration.
  • the controlled release from a biocompatible polymer can be utilized with a water soluble polymer to form an instillable formulation, as well.
  • the controlled release from a biocompatible polymer such as for example, PLGA microspheres or nanospheres, can be utilized in a formulation suitable for intra ocular implantation or injection for sustained release administration, as well any suitable biodegradable and biocompatible polymer can be used.
  • the nucleotide sequence for human ND4 (SEQ ID NO: 6) was obtained based on US National Center for Biotechnology Information reference sequence yp_003024035.1.
  • the sequences for the non-optimized mitochondrial targeting sequence COX10 is SEQ ID NO: 1.
  • the optimized sequences for the mitochondrial targeting sequence COX10 (opt_COX10, SEQ ID NO: 2) and the coding sequence of human ND4 (opt_ND4, SEQ ID NO: 7) were designed to improve the transcription efficiency and the translation efficiency.
  • the optimized COX10-ND4 sequence which is about 75.89% homology to the non-optimized COX10-ND4, was followed by a three prime untranslated region (i.e., 3′UTR, SEQ ID NO: 13) to a recombinant nucleic acid, opt_COX10-opt_ND4-3′UTR (as shown in SEQ ID NO: 31).
  • the pAAV2-opt_ND4 plasmid was compared to the non-optimized pAAV2-ND4 plasmid.
  • the recon screening and identifying steps were similar to the CN102634527B: the plasmid was cultured at 37° C. in a LB plate. Blue colonies and white colonies were appeared, where white colonies were recombinant clones. The white colonies were picked, added to 100 mg/L ampicillin-containing LB culture medium, cultured at 37° C., 200 rpm for 8 hours and then the plasmid were extracted from the cultured bacterial medium based on the Biomiga plasmid extraction protocol. The identification of the plasmid was confirmed using the EcoRI/SalI restriction enzymes.
  • HEK293 cells were inoculated to 225 cm 2 cell culture bottle: at the inoculation density of 3.0 ⁇ 10 7 cells/ml, the culture medium was the Dulbecco's Modified Eagle Medium (DMEM) with 10% bovine serum, at 37° C. in a 5% CO2 incubator overnight. The culture medium were replaced with fresh DMEM with 10% bovine serum on the day of transfection.
  • DMEM Dulbecco's Modified Eagle Medium
  • Virus collection 1) dry ice ethanol bath (or liquid nitrogen) and a 37° C. water bath were prepared; 2) the transfected cells along with media were collected in a 15 ml centrifuge tube, 3) the cells were centrifuged for 3 minutes at 1000 rpm/min; the cells and supernatant were separated, the supernatant were stored separately; and the cells were re-suspended in 1 ml of PBS: 4) the cell suspension were transferred between the dry ice-ethanol bath and 37° C. water bath repeatedly, freeze thawing for four times for 10 minutes each, slightly shaking after each thawing.
  • Virus concentration 1) cell debris were removed with 10,000 g centrifugation; the centrifugal supernatant was transferred to a new centrifuge tube; 2) impurities were removed by filtering with a 0.45 ⁇ m filter; 3) each 1 ⁇ 2 volume of 1M NaCl and 10% PEG 8000 solution were added in the sample, uniformly mixed, and stored at 4° C. overnight; 4) supernatant was discarded after 12,000 rpm centrifugation for 2 h; after the virus precipitate was completely dissolving in an appropriate amount of PBS solution, sterilizing the sample with a 0.22 ⁇ m filter; 5) adding benzonase nuclease was added to remove residual plasmid DNA (final concentration at 50 U/ml). The tube was inverted several times to mix thoroughly and then incubated at 37° C. for 30 minutes; 6) the sample was filtered with a 0.45 ⁇ m filtration head; the filtrate is the concentrated rAAV2 virus.
  • Virus purification 1) CsCl was added to the concentrated virus solution until a density of 1.41 g/ml (refraction index at 1.372): 2) the sample was added to in the ultracentrifuge tube and filled the tube with pre-prepared 1.41 g/ml CsCl solution: 3) centrifuged at 175,000 g for 24 hours to form a density gradient. Sequential collection of different densities of the sample was performed. The enriched rAAV2 particles were collected; 4) repeating the process one more time. The virus was loaded to a 100 kDa dialysis bag and dialyzed/desalted at 4° C. overnight. The concentrated and purified recombinant adeno-associated virus were rAAV2-ND4 and rAAV2-optimized ND4.
  • Virus solution (1-10 1 vg/0.05 mL) was punctured into the vitreous cavity from 3 mm outside the corneal limbus at the pars plana. After the intravitreal injection, the eyes were examined using slit lamp exam and fundus photography inspection. Injection for 30 days. RT-PCR detection and immunoblotting were carried out in each group respectively.
  • RNAs from the transfected rAAV2-ND4 and rAAV2-optimized ND4 rabbit optic nerve cells were extracted using the TRIZOL total RNA extraction kit.
  • cDNA templates were synthesized by reverse transcription of the extracted RNA.
  • NCBI conserved structural domain analysis software were used to analyze the conservative structure of ND4, ensuring that the designed primers amplified fragments were located at non-conserved region; then primers were designed according to the fluorescent quantitative PCR primer design principle:
  • ⁇ -actin-S (SEQ ID NO: 85) CGAGATCGTGCGGGACAT; ⁇ -actin-A: (SEQ ID NO: 86) CAGGAAGGAGGGCTGGAAC; ND4-S: (SEQ ID NO: 87) CTGCCTACGACAAACAGAC; ND4-A: (SEQ ID NO: 88) AGTGCGTTCGTAGTTTGAG;
  • the relative expression level (mRNA level) of the rAAV2-ND4 and rAAV2-optimized ND4 were 0.42 ⁇ 0.23 and 0.57 ⁇ 0.62, respectively (p ⁇ 0.05, FIG. 2 ).
  • the results unexpectedly show that the optimized ND4 (opt_ND4, SEQ ID NO: 7) coding nucleic acid sequence and the corresponding recombinant nucleic acid (opt_COX10-opt_ND4-3′UTR, SEQ ID NO: 31) surprisingly increased the transcription efficiency, increasing the expression of the rAAV2-optimized ND4 by about 36%.
  • the results showed that the transcription efficiency of the rAAV2-optimized ND4 is significantly higher.
  • the ND4 protein was purified from the rabbit nerve cells transfected by rAAV2-optimized ND4 and rAAV2-ND4, respectively. After a 10% polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane (Bio-Rad, HER-hercules, CA, USA) for immune detection. ⁇ -actin was used as an internal reference gene. The film strip was observed on an automatic image analysis instrument (Li-Cor; Lincoln, Nebr., USA) and analyzed using the integrated optical density of the protein band with integral normalization method, so as to obtain the same sample corresponding optical density value. The statistical analysis software SPSS 19.0 was used for the data analysis.
  • the comparison of the two groups is shown in Table 2.
  • the fastest eyesight improving time was 1 month in the experimental group, which was significantly faster than the control group at 3 months (p ⁇ 0.01): the optimal recovery of vision for the experimental group was 1.0, which was obviously higher than the control group at 0.8 (p ⁇ 0.01); the average recovery of vision in the experimental group was 0.582 ⁇ 0.086, which was obviously higher than the control group at 0.344 ⁇ 0.062 (p ⁇ 0.01).
  • the fundus photographic results were shown in FIG. 5 . No obvious damage or complication to the optic nerve and retinal vascular of the patients in the experimental and control groups, indicating the safety of the intravitreal injection of rAAV2-optimized ND4 and rAAV2-ND4.
  • COX10 and 3′UTR sequences in the recombinant nucleic acid (opt_COX10-opt_ND4-3′UTR, SEQ ID NO: 31) in examples 1-6 were replaced with another mitochondrial targeted sequence, OPA1 (SEQ ID NO: 5) and another 3′UTR sequence, 3′UTR* (SEQ ID NO: 14) respectively, to generate a new recombinant nucleic acid, OPA1-opt_ND4-3′UTR* (SEQ ID NO: 74).
  • the frozen 293T cell was resuscitated and allowed to grow in a T75 flask to about 90%.
  • the cells were precipitated and resuspended in DMEM complete medium to a cell density of 5 ⁇ 10 4 cells/mL.
  • the cells were resuspended.
  • About 100 ⁇ l of the cell suspension (about 5000 cells) were added in each well of a 96 well plate.
  • the cells were cultured and grown to 50% under 37° C. and 5% CO 2 .
  • About 0.02 ⁇ l PBS was mixed with 2 ⁇ 10 ⁇ l vg/0.02 ⁇ l of the virus rAAV2-ND4-EGFP and rAAV2-opt_ND4*-EGFP, respectively.
  • ⁇ -actin-S (SEQ ID NO: 85) CGAGATCGTGCGGGACAT; ⁇ -actin-A: (SEQ ID NO: 86) CAGGAAGGAGGGCTGGAAC; ND4-S: (SEQ ID NO: 107) GCCAACAGCAACTACGAGC; ND4-A: (SEQ ID NO: 108) TGATGTTGCTCCAGCTGAAG;
  • FIG. 7 shows the ND4 expression in 293T cells.
  • the average expression of ND4 protein for rAAV2-ND4 is 0.36, while the average expression of ND4 protein for rAAV2-opt_ND4* is 1.65, which is about 4.6 times higher than the rAAV2-ND4 group (p ⁇ 0.01) (see FIG. 8 ).
  • FIG. 9 shows the ND4 expression in rabbit optic nerve cells.
  • the average expression of ND4 protein for rAAV2-ND4 is 0.16, while the average expression of ND4 protein for rAAV2-opt_ND4* is 0.48, which is about 3 times higher than the rAAV2-ND4 group (p ⁇ 0.01) (see FIG. 10 ).
  • Eye balls from both rabbit groups were removed after the slit lamp examination and intraocular pressure measurement. Eye balls were fixed, and dehydrated using paraffin. Tissues were pathologically sectioned along the direction of optic nerves. After further dehydration, the tissue sample was dyed using hematoxylin and eosin. The microscope inspection result is referred to FIG. 12 . As shown in the HE staining results, the rabbit retinal ganglion fiber layer was not damaged and the number of ganglion cells was not reduced, indicating the intravitreal injection did not produce retinal toxicity or nerve damage, and can be used safely.
  • COX10-ND6-3′UTR SEQ ID NO: 21
  • SEQ ID NO: 21 is the combination (5′ to 3′) of COX10 (SEQ ID NO: 1), ND6 (SEQ ID NO: 9), and 3′UTR (SEQ ID NO: 13).
  • ND6-F (SEQ ID NO: 89) ATGATGTATGCTTTGTTTCTG
  • ND6-R (SEQ ID NO: 90) CTAATTCCCCCGAGCAATCTC
  • the transfected and screened virus rAAV2-ND6 had a viral titer of 2.0 ⁇ 10 11 vg/mL. Similar to example 5, slit lamp examination and intraocular pressure measurement was performed on three groups of rabbits (A: rAAV2-ND6; B: rAAV-GFP; C: PBS) at 1, 7, and 30 days after the surgery ( FIG. 13 ). No obviously abnormality, conjunctival congestion, secretions, or endophthalmitis were observed and the intraocular pressure were not elevated in all the rabbits.
  • ⁇ -actin-S (SEQ ID NO: 85) CGAGATCGTGCGGGACAT; ⁇ -actin-A: (SEQ ID NO: 86) CAGGAAGGAGGGCTGGAAC; ND6-S: (SEQ ID NO: 91) AGTGTGGGTTTAGTAATG; ND4-A: (SEQ ID NO: 92) TGCCTCAGGATACTCCTC;
  • FIG. 14 shows the fundus photographic results for rabbits injected with rAAV2-opt_ND6 (A), rAAV2-ND6 (B), rAAV-EGFP (C), respectively. No obviously abnormality, conjunctival congestion, secretions, or endophthalmitis were observed and the intraocular pressure were not elevated in all the rabbits.
  • ⁇ -actin-F (SEQ ID NO: 93) CTCCATCCTGGCCTCGCTGT; ⁇ -actin-R: (SEQ ID NO: 94) GCTGTCACCTTCACCGTTCC; ND6-F: (SEQ ID NO: 95) GGGTTTTCTTCTAAGCCTTCTCC; ND6-R: (SEQ ID NO: 96) CCATCATACTCTTTCACCCACAG; opt_ND6-F: (SEQ ID NO: 97) CGCCTGCTGACCGGCTGCGT; opt_ND6-R: (SEQ ID NO: 98) CCAGGCCTCGGGGTACTCCT;
  • ND1-F (SEQ ID NO: 99) ATGGCCGCATCTCCGCACACT, ND1-R: (SEQ ID NO: 100) TTAGGTTTGAGGGGGAATGCT,
  • the plasmid screening for opt_COX10*-opt_ND1-3′UTR (SEQ ID NO: 55) used the following primers:
  • ND1-F (SEQ ID NO: 101) AACCTCAACCTAGGCCTCCTA, ND1-R: (SEQ ID NO: 102) TGGCAGGAGTAACCAGAGGTG,
  • B 10 10 vg/50 ⁇ l of rAAV2-ND1
  • C 10 10 vg/50 ⁇ l of rAAV2-EGFP. No obviously abnormality, conjunctival congestion, secretions, or endophthalmitis were observed and the intraocular pressure were not elevated in all the rabbits.
  • ND1-F (SEQ ID NO: 103) AGGAGGCTCTGTCTGGTATCTTG; ND1-R: (SEQ ID NO: 104) TTTTAGGGGCTTCTTTGGTGAA; opt_ND1-F: (SEQ ID NO: 105) GCCGCCTGCTGACCGGCTGCGT; opt_ND1-R: (SEQ ID NO: 106) TGATGTACAGGGTGATGGTGCTGG;
  • AAV2 virus samples were used to screen different AAV formulations.
  • the stability of the different AAV formulations were evaluated using the StepOnePlus real-time PCR system.
  • the viral titer of each formulation under a freeze/thaw cycle condition was measured.
  • formulations tested were: A: phosphate-buffered saline (PBS); B: 1% ⁇ , ⁇ -trehalose dehydrate, 1% L-histidine monohydrochloride monohydrate, and 1% polysorbate 20; and C: 180 mM NaCl, 10 mM NaH 2 PO 4 /Na 2 HPO 4 , and 0.001% poloxamer 188, pH 7.3.
  • PBS phosphate-buffered saline
  • B 1% ⁇ , ⁇ -trehalose dehydrate, 1% L-histidine monohydrochloride monohydrate, and 1% polysorbate 20
  • C 180 mM NaCl, 10 mM NaH 2 PO 4 /Na 2 HPO 4 , and 0.001% poloxamer 188, pH 7.3.
  • RSD relative standard deviation
  • formulation C has the lowest relative standard deviation (RSD) after 5 freeze/thaw cycles, indicating superior stability as an AAV formulation.
  • D phosphate-buffered saline
  • E PBS and 0.001% poloxamer 188, pH 7.2-7.4
  • F 80 mM NaCl, 5 mM NaH 2 PO 4 , 40 mM Na 2 HPO 4 , 5 mM KH 2 PO 4 and 0.001% poloxamer 188, 7.2-7.4.
  • formulation F has the lowest relative standard deviation (RSD) after 5 freeze/thaw cycles, indicating superior stability as an AAV formulation. Overall, formulation F also has the lowest RSD among all tested formulations and can be used as the AAV formulation for future development.
  • RSD relative standard deviation

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Abstract

Disclosed herein is a recombinant nucleic acid, comprising: a mitochondrial targeting sequence; a mitochondrial protein coding sequence, wherein said mitochondrial protein coding sequence encodes a polypeptide comprising a mitochondrial protein; and a 3′UTR nucleic acid sequence. Also disclosed is a pharmaceutical composition comprising the recombinant nucleic acid and a method of treating Leber's hereditary optic neuropathy (LHON) using the pharmaceutical composition.

Description

    CROSS-REFERENCE
  • This application claims the benefit of PCT Application No. PCT/CN2018/095023, filed on Jul. 9, 2018; PCT Application No. PCT/CN2018/103937, filed on Sep. 4, 2018; Chinese Application Nos. CN201810703168.7 and CN201810702492.7, both filed on Jun. 29, 2018; PCT Application No. PCT/CN2018/113799, filed on Nov. 2, 2018; Chinese Application No. CN201811230856.2, filed on Oct. 22, 2018; PCT Application No. PCT/CN2018/118662, filed on Nov. 30, 2018; Chinese Application No. CN201811221305.X, filed on Oct. 19, 2018; PCT Application No. PCT/CN2019/070461, filed on Jan. 4, 2019; Chinese Application No. CN201810948193.1, filed on Aug. 20, 2018; all of which are incorporated herein by reference in their entirety.
    • REFERENCE TO A SEQUENCE LISTING
  • The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 30, 2019, is named 207298476_1.txt and is 304,914 bytes in size.
    • BACKGROUND OF THE INVENTION
  • Lebers hereditary optic neuropathy (LHON) is a mitochondrially inherited (transmitted from mother to offspring) degeneration of retinal ganglion cells (RGCs) and their axons that leads to an acute or subacute loss of central vision; this affects predominantly young adult males. LHON is only transmitted through the mother, as it is primarily due to mutations in the mitochondrial (not nuclear) genome, and only the egg contributes mitochondria to the embryo. LHON is usually due to one of three pathogenic mitochondrial DNA (mtDNA) point mutations. These mutations are at nucleotide positions 11778 G to A (G11778A), 3460 G to A (G3460A) and 14484 T to C (TI 4484C), respectively in the NADH dehydrogenase subunit-4 protein (ND4), NADH dehydrogenase subunit-1 protein (ND1) and NADH dehydrogenase subunit-6 protein (ND6) subunit genes of complex I of the oxidative phosphorylation chain in mitochondria. Each mutation is believed to have significant risk of permanent loss of vision. It typically progresses within several weeks to several months without pain, until the binocular vision deteriorate to below 0.1, which seriously affects the quality of life of the patient. Two LHON mutants, G3460A and T14484C, results in the reduction of the patient's platelets isolated mitochondrial NADH dehydrogenase activity by 80%. Ninety percent of the Chinese LHON patients carry the G11778A mutation. The G11778A mutation changes an arginine into histidine in the ND4 protein, resulting the dysfunction and optic nerve damage in LHON patients. There is a need for developing compositions and methods for treating LHON with higher transfection efficiency and treatment efficacy.
    • SUMMARY OF THE INVENTION
  • Disclosed here recombinant nucleic acids, pharmaceutical compositions, and methods for treating LHON. In one aspect, disclosed herein is a recombinant nucleic acid, comprising: a mitochondrial targeting sequence; a mitochondrial protein coding sequence comprising a sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NO: 7, 8, 10, and 12; and a 3′UTR nucleic acid sequence.
  • In some cases, the mitochondrial targeting sequence encodes a polypeptide comprising a peptide sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 129-159. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 2. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 3. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 4. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 5.
  • In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 7 or 8. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 10. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 12.
  • In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 111-125. In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14.
  • In some cases, the recombinant nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 17-20, 23-24, 27-28, 31-34, 37-38, 41-42, 45-48, 51-52, 55-56, 59-62, 65-66, 69-70, 73-76, 79-80, and 83-84.
  • In another aspect, disclosed herein is a recombinant nucleic acid, comprising: a mitochondrial targeting sequence comprising a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, and 5; a mitochondrial protein coding sequence, wherein the mitochondrial protein coding sequence encodes a polypeptide comprising a mitochondrial protein; and a 3′UTR nucleic acid sequence.
  • In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 2. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 3. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 4. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 5.
  • In some cases, the mitochondrial protein is selected from a group consisting of NADH dehydrogenase 4 (ND4), NADH dehydrogenase 6 (ND6), NADH dehydrogenase 1 (ND1), and a variant thereof. In some cases, the mitochondrial protein comprises NADH dehydrogenase 4 (ND4), or a variant thereof. In some cases, the mitochondrial protein comprises a peptide sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 160. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 6, 7, or 8. In some cases, the mitochondrial protein comprises NADH dehydrogenase 6 (ND6), or a variant thereof. In some cases, the mitochondrial protein comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 161. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 9 or 10. In some cases, the mitochondrial protein comprises NADH dehydrogenase 1 (ND1), or a variant thereof. In some cases, the mitochondrial protein comprises a sequence that is at least 90/o, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 162. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 11 or 12.
  • In some cases, the 3′UTR nucleic acid sequence is located at 3′ of the mitochondrial targeting sequence. In some cases, the 3′UTR nucleic acid sequence comprises a sequence selected from the group consisting of hsACO2, hsATP5B, hsAK2, hsALDH2, hsCOX10, hsUQCRFS1, hsNDUFV1, hsNDUFV2, hsSOD2, hsCOX6c, hsIRPl, hsMRPS12, hsATP5J2, mSOD2, and hsOXA1L. In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 111-125. In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14.
  • In some cases, the mitochondrial targeting sequence is located at 5′ of the 3′UTR nucleic acid sequence. In some cases, the mitochondrial targeting sequence is located at 3′ of the mitochondrial targeting sequence.
  • In some cases, the recombinant nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 29-84.
  • In another aspect, disclosed herein is a recombinant nucleic acid, comprising: a mitochondrial targeting sequence; a mitochondrial protein coding sequence comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 7, 8, 10, and 12; and a 3′UTR nucleic acid sequence.
  • In some cases, the mitochondrial targeting sequence comprises a sequence encodes a polypeptide selected from the group consisting of hsCOX10, hsCOX8, scRPM2, lcSirt5, tbNDUS7, ncQCR2, hsATP5G2, hsLACTB, spilv1, gmCOX2, crATP6, hsOPA1, hsSDHD, hsADCK3, osP0644B06.24-2, Neurospora crassa ATP9 (ncATP9), hsGHITM, hsNDUFAB1, hsATP5G3, crATP6_hsADCK3, ncATP9_ncATP9, zmLOC100282174, ncATP9_zmLOC100282174_spilv1_ncATP9, zmLOC100282174_hsADCK3_crATP6_hsATP5G3, zmLOCIO0282174_hsADCK3_hsATP5G3, ncATP9_zmLOC100282174, hsADCK3 zmLOC100282174 crATP6 hsATP5G3, crATP6_hsADCK3_zmLOC100282174_hsATP5G3, hsADCK3_zmLOC100282174, hsADCK3_zmLOC100282174_crATP6, ncATP9_zmLOC100282174_spilv1_GNFP_ncATP9, and ncATP9_zmLOC100282174_spilv1_cSirtS_osP0644B06.24-2_hsATP5G2_ncATP9. In some cases, the mitochondrial targeting sequence encodes a polypeptide comprising a peptide sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 129-159. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 2 or 3. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 4. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 5.
  • In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 7 or 8. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 10. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 12.
  • In some cases, the 3′UTR nucleic acid sequence is located at 3′ of the mitochondrial targeting sequence. In some cases, the 3′UTR nucleic acid sequence comprises a sequence selected from the group consisting of hsACO2, hsATPSB, hsAK2, hsALDH2, hsCOX10, hsUQCRFS1, hsNDUFV1, hsNDUFV2, hsSOD2, hsCOX6c, hs1RP1, hsMRPS12, hsATP5J2, mSOD2, and hsOXA1L. In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 111-125. In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14.
  • In some cases, the mitochondrial targeting sequence is located at 5′ of the 3′UTR nucleic acid sequence. In some cases, the mitochondrial targeting sequence is located at 3′ of the mitochondrial targeting sequence.
  • In some cases, the recombinant nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 17-20, 23-24, 27-28, 31-34, 37-38, 41-42, 4548, 51-52, 55-56, 59-62, 65-66, 69-70, 73-76, 79-80, and 83-84.
  • In another aspect, disclosed herein is a recombinant nucleic acid, comprising a mitochondrial targeting sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 2, 3, and 4. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 2. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 3. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 4.
  • In some cases, the recombinant nucleic acid further comprises a mitochondrial protein coding sequence, wherein the mitochondrial protein coding sequence encodes a polypeptide comprising a mitochondrial protein. In some cases, the mitochondrial protein is selected from a group consisting of NADH dehydrogenase 4 (ND4), NADH dehydrogenase 6 (ND6), NADH dehydrogenase 1 (ND1), and a variant thereof. In some cases, the mitochondrial protein comprises NADH dehydrogenase 4 (ND4), or a variant thereof. In some cases, the mitochondrial protein comprises a peptide sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 160. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 6, 7, or 8. In some cases, the mitochondrial protein comprises NADH dehydrogenase 6 (ND6), or a variant thereof. In some cases, the mitochondrial protein comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 161. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 9 or 10. In some cases, the mitochondrial protein comprises NADH dehydrogenase 1 (ND1), or a variant thereof. In some cases, the mitochondrial protein comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 162. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 11 or 12.
  • In some cases, the recombinant nucleic acid further comprises a 3′UTR nucleic acid sequence. In some cases, the 3′UTR nucleic acid sequence is located at 3′ of the mitochondrial targeting sequence. In some cases, the 3′UTR nucleic acid sequence comprises a sequence selected from the group consisting of hsACO2, hsATP5B, hsAK2, hsALDH2, hsCOX10, hsUQCRFS1, hsNDUFV1, hsNDUFV2, hsSOD2, hsCOX6c, hsIRPl, hsMRPS12, hsATP5J2, mSOD2, and hsOXA1L. In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 111-125. In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14. In some cases, the mitochondrial targeting sequence is located at 5′ of the 3′UTR nucleic acid sequence. In some cases, the mitochondrial targeting sequence is located at 3′ of the mitochondrial targeting sequence.
  • In some cases, the recombinant nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 29-70.
  • In another aspect, disclosed herein is a recombinant nucleic acid, comprising a mitochondrial protein coding sequence, wherein the mitochondrial protein coding sequence encodes a polypeptide comprising a mitochondrial protein, wherein the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 7, 8, 10, and 12.
  • In some cases, the recombinant nucleic acid further comprises a mitochondrial targeting sequence. In some cases, the mitochondrial targeting sequence comprises a sequence encodes a polypeptide selected from the group consisting of hsCOX10, hsCOX8, scRPM2, lcSirt5, tbNDUS7, ncQCR2, hsATP5G2, hsLACTB, spilv1, gmCOX2, crATP6, hsOPA1, hsSDHD, hsADCK3, osP0644B06.24-2, Neurospora crassa ATP9 (ncATP9), hsGHITM, hsNDUFAB1, hsATP5G3, crATP6_hsADCK3, ncATP9_ncATP9, zmLOC100282174, ncATP9_zmLOC100282174_spily i_ncATP9, zmLOC100282174_hsADCK3_crATP6_hsATP5G3, zmLOC100282174_hsADCK3_hsATP5G3, ncATP9_zmLOC100282174, hsADCK3_zmLOC100282174_crATP6_hsATP5G3, crATP6_hsADCK3_zmLOC100282174_hsATP5G3, hsADCK3_zmLOC100282174, hsADCK3_zmLOC100282174_crATP6, ncATP9_zmLOC100282174_spilv1_GNFP_ncATP9, and ncATP9_zmLOC100282174_spilv1_lcSirt5_osP0644B06.24-2_hsATP5G2_ncATP9. In some cases, the mitochondrial targeting sequence encodes a polypeptide comprising a peptide sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 129-159. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 2. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 3. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 4. In some cases, the mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 5.
  • In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 7 or 8. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 10. In some cases, the mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97/a, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 12.
  • In some cases, the recombinant nucleic acid further comprises a3′UTR nucleic acid sequence. In some cases, the 3′UTR nucleic acid sequence is located at 3′ of the mitochondrial targeting sequence. In some cases, the 3′UTR nucleic acid sequence comprises a sequence selected from the group consisting of hsACO2, hsATPSB, hsAK2, hsALDH2, hsCOX10, hsUQCRFS1, hsNDUFV1, hsNDUFV2, hsSOD2, hsCOX6c, hslRP1, hsMRPS12, hsATP5J2, mSOD2, and hsOXA1L. In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 111-125. In some cases, the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14. In some cases, the mitochondrial targeting sequence is located at 5′ of the 3′UTR nucleic acid sequence. In some cases, the mitochondrial targeting sequence is located at 3′ of the mitochondrial targeting sequence.
  • In some cases, the recombinant nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 17-20, 23-24, 27-28, 31-34, 37-38, 41-42, 45-48, 51-52, 55-56, 59-62, 65-66, 69-70, 73-76, 79-80, and 83-84.
  • In another aspect, disclosed herein is a viral vector comprising the recombinant nucleic acid disclosed herein. In some cases, the viral vector is an adeno-associated virus (AAV) vector. In some cases, the AAV vector is selected from the group consisting ofAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16 vectors. In some cases, the AAV vector is a recombinant AAV (rAAV) vector. In some cases, the rAAV vector is rAAV2 vector.
  • In another aspect, disclosed herein is a pharmaceutical composition, comprising an adeno-associated virus (AAV) comprising any recombinant nucleic acid disclosed herein. In some cases, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient thereof. Also disclosed is a pharmaceutical composition, comprising the viral vector disclosed herein, and a pharmaceutically acceptable excipient thereof, wherein the viral vector comprises any recombinant nucleic acid disclosed herein. Also disclosed is a pharmaceutical composition, comprising: an adeno-associated virus (AAV) comprising any recombinant nucleic acid disclosed herein, wherein the recombinant nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 15; and a pharmaceutically acceptable excipient.
  • In some cases, the pharmaceutically acceptable excipient comprises phosphate-buffered saline (PBS), α,α-trehalose dehydrate, L-histidine monohydrochloride monohydrate, polysorbate 20, NaCl, NaH2PO4. Na2HPO4, KH2PO4, K2HPO4, poloxamer 188, or any combination thereof. In some cases, the pharmaceutically acceptable excipient is selected from phosphate-buffered saline (PBS), α,α-trehalose dehydrate, L-histidine monohydrochloride monohydrate, polysorbate 20, NaCl, NaH2PO4, Na2HPO4, KH2PO4, K2HPO4, poloxamer 188, and any combination thereof. In some cases, the pharmaceutically acceptable excipient comprises poloxamer 188. In some cases, the pharmaceutically acceptable excipient comprises 0.0001%-0.01% poloxamer 188. In some cases, the pharmaceutically acceptable excipient comprises 0.001% poloxamer 188. In some cases, the pharmaceutically acceptable excipient further comprises one or more salts. In some cases, the one or more salts comprises NaCl, NaH2PO4, Na2HPO4, and KH2PO4. In some cases, the one or more salts comprises 80 mM NaCl, 5 mM NaH2PO4, 40 mM Na2HPO4, and 5 mM KH2PO4. In some cases, the pharmaceutical composition has a pH of 6-8. In some cases, the pharmaceutical composition has a pH of 7.2-7.4. In some cases, the pharmaceutical composition has a pH of 7.3. In some cases, the pharmaceutical composition has a viral titer of at least 1.0×1010 vg/mL. In some cases, the pharmaceutical composition has a viral titer of at least 5.0×1010 vg/mL.
  • In some cases, the pharmaceutical composition is subject to five freeze/thaw cycles, the pharmaceutical composition retains at least 60%, 70%, 80%, or 90% of a viral titer as compared to the viral titer prior to the five freeze/thaw cycles. In some cases, the pharmaceutical composition, when administered to a patient with Leber's hereditary optic neuropathy, generates a higher average recovery of vision than a comparable pharmaceutical composition without the recombinant nucleic acid. In some cases, the pharmaceutical composition, when administered to a patient with Leber's hereditary optic neuropathy, generates a higher average recovery of vision than a comparable pharmaceutical composition comprising a recombinant nucleic acid as set forth in SEQ ID NO: 15.
  • In another aspect, disclosed herein is a method of treating an eye disorder, comprising administering any pharmaceutical composition disclosed herein to a patient in need thereof. In some cases, the eye disorder is Leber's hereditary optic neuropathy (LHON). In some cases, the method comprises administering the pharmaceutical composition to one or both eyes of the patient. In some cases, the pharmaceutical composition is administered via intraocular or intravitreal injection. In some cases, the pharmaceutical composition is administered via intravitreal injection. In some cases, about 0.01-0.1 mL of the pharmaceutical composition is administered via intravitreal injection. In some cases, about 0.05 mL of the pharmaceutical composition is administered via intravitreal injection.
  • In some cases, the method further comprises administering methylprednisolone to the patient. In some cases, the methylprednisolone is administered prior to the intravitreal injection of the pharmaceutical composition. In some cases, the methylprednisolone is administered orally In some cases, the methylprednisolone is administered daily for at least 1, 2, 3, 4, 5, 6, or 7 days prior to the intravitreal injection of the pharmaceutical composition. In some cases, the methylprednisolone is administered daily. In some cases, the a daily dosage of about 32 mg/60 kg methylprednisolone is administered. In some cases, the methylprednisolone is administered after the intravitreal injection of the pharmaceutical composition. In some cases, the method further comprises administering creatine phosphate sodium to the patient. In some cases, the creatine phosphate sodium is administered intravenously. In some cases, the methylprednisolone is administered intravenously or orally. In some cases, the method comprises administering methylprednisolone intravenously for at least one day, which is followed by administering methylprednisolone orally for at least a week. In some cases, the method comprises administering methylprednisolone intravenously for about 3 days, which is followed by administering methylprednisolone orally for at least about 6 weeks. In some cases, the methylprednisolone is administered intravenously at a daily dose of about 80 mg/60 kg. In some cases, the administering the pharmaceutical composition generates a higher average recovery of vision than a comparable pharmaceutical composition without the recombinant nucleic acid. In some cases, the administering the pharmaceutical composition generates a higher average recovery of vision than a comparable pharmaceutical composition comprising a recombinant nucleic acid as set forth in SEQ ID NO: 15.
    • INCORPORATION BY REFERENCE
  • All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
  • FIG. 1 shows the PCR nucleic acid electrophoresis verification of ND4 (lane A) and optimized ND4 (lane B) gene cloning results.
  • FIG. 2 shows the relative expression level comparison using qPCR between the rAAV2-opt_ND4 (left black column) and rAAV2-ND4 (right black column). β-actin is the internal reference gene (white column).
  • FIG. 3 shows the relative expression level comparison using immunoblotting between the rAAV2-opt_ND4 (left black column) and rAAV2-ND4 (right black column). β-actin is the internal reference gene (white column).
  • FIG. 4 shows the fundus photographic results for rabbits injected with rAAV2-opt_ND4 (right) and rAAV2-ND4 (left), respectively.
  • FIG. 5 shows the fundus photographic results for a patient before (left) and after (right) the injection with rAAV2-optimized ND4.
  • FIG. 6 shows EGFP expression levels of rAAV2-ND4 (left) and rAAV2-opt_ND4* (right).
  • FIG. 7 shows the ND4 expression in 293T cells: rAAV2-ND4 (left) and rAAV2-opt_ND4* (right).
  • FIG. 8 shows the relative ND4 expression in 293T cells: rAAV2-ND4 (left) and rAAV2-opt_ND4* (right).
  • FIG. 9 shows the ND4 expression in rabbit optic nerve cells: rAAV2-ND4 (left) and rAAV2-opt_ND4* (right).
  • FIG. 10 shows the relative ND4 expression in rabbit optic nerve cells: rAAV2-ND4 (left) and rAAV2-opt_ND4* (right).
  • FIG. 11 shows the fundus photographic results for rAAV2-ND4 (left) and rAAV2-opt_ND4* (right).
  • FIG. 12 shows the microscope inspection (HE staining) results for rAAV2-ND4 (left) and rAAV2-opt_ND4* (right).
  • FIG. 13 shows the fundus photographic results for rabbits injected with rAAV2-ND6 (A), rAAV-GFP (B) and PBS, respectively.
  • FIG. 14 shows the fundus photographic results for rabbits injected with rAAV2-opt_ND6 (A), rAAV2-ND6 (B), rAAV-EGFP (C), respectively.
  • FIG. 15 shows the relative ND6 expression in rabbit optic nerve cells: rAAV2-opt_ND6 (A), rAAV2-ND6 (B), and rAAV-EGFP (C).
  • FIG. 16 shows the relative ND6 expression by western blot: rAAV2-opt_ND6 (A), rAAV2-ND6 (B), and rAAV-EGFP (C).
  • FIG. 17 shows the relative ND1 expression in rabbit optic nerve cells: rAAV2-opt_ND1 (A), rAAV2-ND1 (B), and rAAV-EGFP (C).
  • FIG. 18 shows the relative ND1 expression by western blot: rAAV2-opt_ND1 (A), rAAV2-ND1 (B), and rAAV-EGFP (C).
    •  DETAILED DESCRIPTION OF THE INVENTION Definitions
  • Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies. The materials, methods and examples are illustrative only and not limiting.
  • As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes a plurality of such agents, and reference to “the salt” includes reference to one or more salts (or to a plurality of salts) and equivalents thereof known to those skilled in the art, and so forth.
  • As used herein, unless otherwise indicated, the term “or” can be conjunctive or disjunctive. As used herein, unless otherwise indicated, any embodiment can be combined with any other embodiment.
  • As used herein, unless otherwise indicated, some inventive embodiments herein contemplate numerical ranges. When ranges are present, the ranges include the range endpoints. Additionally, every subrange and value within the range is present as if explicitly written out.
  • The term “about” and its grammatical equivalents in relation to a reference numerical value and its grammatical equivalents as used herein can include a range of values plus or minus 10% from that value, such as a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. For example, the amount “about 10” includes amounts from 9 to 11.
  • The term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including”) is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein, may “consist of” or “consist essentially of” the described features.
  • The term “subject” refers to a mammal that has been or will be the object of treatment, observation or experiment. The term “mammal” is intended to have its standard meaning, and encompasses humans, dogs, cats, sheep, and cows, for example. The methods described herein can be useful in both human therapy and veterinary applications. In some embodiments, the subject is a human.
  • The term “treating” or “treatment” encompasses administration of at least one compound disclosed herein, or a pharmaceutically acceptable salt thereof, to a mammalian subject, particularly a human subject, in need of such an administration and includes (i) arresting the development of clinical symptoms of the disease, such as cancer, (ii) bringing about a regression in the clinical symptoms of the disease, such as cancer, and/or (iii) prophylactic treatment for preventing the onset of the disease, such as cancer.
  • The term “therapeutically effective amount” of a chemical entity described herein refers to an amount effective, when administered to a human or non-human subject, to provide a therapeutic benefit such as amelioration of symptoms, slowing of disease progression, or prevention of disease.
  • As used herein, unless otherwise indicated, the terms “nucleic acid” and “polynucleotide” can be used interchangeably.
    • Nucleic Acid and Polypeptide Sequences
  • Table 1 discloses all the nucleic acid and polypeptide sequences disclosed herein. The first column shows the SEQ ID NO of each sequence. The second column describes the nucleic acid or polypeptide construct. For example, the construct COX10-ND6-3′UTR is a nucleic acid combining the nucleic acid sequences of COX10 (SEQ ID NO: 1), ND6 (SEQ ID NO: 9), and 3′UTR (SEQ ID NO: 13) (from 5′ to 3′ without linker between the nucleic acid sequences.
  • TABLE 1
    nucleic acid and polypeptide sequences and SEQ ID NOs
    SEQ description sequence
    1 COX10 ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAGAACT
    2 opt_COX10 ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACA
    3 opt_COX10* ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACC
    4 COX8 ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTG
    5 CPA1 GTGCTGCCCGCCTAGAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGQTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTG
    6 ND4 ATGCTAAAACTAATCGTCCCAACATTTATGTTACTACCACTGACATGGCTTTCCAAAAAACACATGATTT
    GGATCAACACAACCACCCACAGCCTAATTATTAGCATCATCCCTCTACTATTTTTTAACCAAATCAACAA
    CAACCTATTTAGCTGTTCCCCAACCTTTTCCTCCGACCCCCTAACAACCCCCCTCCTAATGCTAACTAC
    CTGGCTCCTACCCCTCACAATCATGGCAAGCCAAGGCCACTTATCCAGTGAACCACTATCACGAAAAA
    AACTCTACCTCTCTATGCTAATCTCCCTACAAATCTCCTTAATTATGACATTCACAGCCACAGAACTAAT
    CATGTTTTATATCTTCTTCGAAACCACACTTATCCCCACCTTGGCTATCATCACCCGATGGGGCAACCA
    GCCAGAACGCCTGAACGCAGGCACATACTTCCTATTCTACACCCTAGTAGGCTCCCTTCCCCTACTCA
    TCGCACTAATTTACACTCACAACACCCTAGGCTCACTAAACATTCTACTACTCACTCTGACTGCCCAAG
    AACTATCAAACTCCTGGGCCAACAACTTAATGTGGCTAGCTTACACAATGGCTTTTATGGTAAAGATGC
    CTCTTTACGGACTCCACTTATGGCTCCCTAAAGCCCATGTCGAAGCCCCCATCGCTGGGTCAATGGTA
    CTTGCCGCAGTACTCTTAAAACTAGGCGGCTATGGTATGATGCGCCTCACACTCATTCTCAACCCCCT
    GACAAAACACATGGCCTACCCCTTCCTTGTACTATCCCTATGGGGCATGATTATGACAAGCTCCATCTG
    CCTACGAGAAACAGACCTAAAATCGCTCATTGCATACTCTTCAATGAGCCACATGGCCCTCGTAGTAAC
    AGCCATTCTCATCCAAACCCCCTGGAGCTTCACCGGCGCAGTCATTCTCATGATCGCCCACGGGCTTA
    CATCCTCATTACTATTCTGCCTAGCAAACTCAAACTACGAACGCACTCACAGTCGCATCATGATCCTCT
    CTCAAGGACTTCAAACTCTACTCCCACTAATGGCTTTTTGGTGGCTTCTAGCAAGCCTCGCTAACCTCG
    CCTTACCCCCCACTATTAACCTACTGGGAGAACTGTCTGTGQTAGTAACCACGTTCTCCTGGTGAAATA
    TCACTCTCCTACTTACAGGACTCAACATGCTAGTCACAGCCCTATACTCCCTCTACATGTTTACCACAA
    CACAATGGGGCTCACTCACCCACCACATTAACAACATGAAACCCTCATTCACACGAGAAAACACCCTC
    ATGTTCATGCACCTATCCCCCATTCTCCTCCTATCCCTCAACCCCGACATCATTACCGGGTTTTCCTCT
    TAA
    7 opt_ND4 ATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCTCTGACCTGGCTGAGCAAGAAACACATGAT
    CTGGATCAACACCACCACGCACAGCCTGATCATGAGCATCATCCGTGTGCTGTTCTTCAACCAGATCA
    ACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCTCTGACAACACCTCTGCTGATGCTG
    ACCACCTGGCTGCTGCCCCTCACAATCATGGCCTCTCAGAGACACCTGAGCAGCGAGCCCCTGAGCC
    GGAAGAAACTGTACCTGAGCATGCTGATCTCCCTGCAGATCTCTCTGATCATGACCTTCACCGCCACC
    GAGCTGATCATGTTQTAGATCTTTTTCGAGAGAACGCTGATCCGCACACTGGCCATCATCACCAGATG
    GGGCAACCAGCCTGAGAGACTGAACGCCGGCACCTACTTTCTGTTCTACACCCTCGTGGGCAGCCTG
    CCACTGCTGATTGCCCTGATCTACACCCACAACACCCTGGGCTCCCTGAACATCCTGCTGCTGACACT
    GACAGCCCAAGAGCTGAGCAACAGCTGGGCCAACAATCTGATGTGGCTGGCCTACACAATGGCCTTC
    ATGGTCAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCTAAAGCTCATGTGGAAGCCCCTATCG
    CCGGCTCTATGGTGCTGGCTGCAGTGCTGCTGAAACTCGGCGGCTACGGCATGATGCGGCTGACCCT
    GATTCTGAATCCCCTGACCAAGCACATGGCCTATCCATTTCTGGTGCTGAGCCTGTGGGGCATGATTA
    TGACCAGCAGCATCTGCCTGCGGCAGACCGATCTGAAGTCCCTGATCGCCTACAGCTCCATCAGCCA
    CATGGCCCTGGTGGTCACCGCCATCCTGATTCAGACCCCTTGGAGCTTTACAGGCGCCGTGATCCTG
    ATGATTGCCCACGGCCTGACAAGGAGCCTGGTGTTTTGTCTGGCCAACAGCAACTAGGAGCGGACCC
    ACAGCAGAATCATGATCCTGTCTCAGGGCCTGCAGACCCTCCTGCCTCTTATGGCTTTTTGGTGGCTG
    CTGGCCTCTCTGGCCAATCTGGCACTGCCTCCTACCATCAATCTGCTGGGCGAGCTGAGCGTGCTGG
    TCACCACATTCAGCTGGTCCAATATCACCCTGCTGCTCACCGGCCTGAACATGCTGGTTACAGCCCTG
    TACTCCCTGTACATGTTCACCACCACACAGTGGGGAAGCCTGAGACACCACATCAACAATATGAAGCC
    CAGCTTCACCCGCGAGAACACCCTGATGTTCATGCATCTGAGCCCCATTCTGCTGCTGTCCCTGAATC
    CTGATATCATCACCGGCTTCTCCAGCTGA
    8 opt_ND4* ATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCCCTGACCTGGCTGAGCAAGAAGCACATGA
    TCTGGATCAACACCACCACCCACAGCCTGATCATCAGCATCATCCCCCTGCTGTTCTTCAACCAGATC
    AACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCCCTGACCACCCCCCTGCTGATGC
    TGACCACCTGGCTGCTGCCCCTGACCATCATGGCCAGCCAGCGCCACCTGAGCAGCGAGCCCCTGA
    GCCGCAAGAAGCTGTACCTGAGCATGCTGATCAGCCTGCAGATCAGCCTGATCATGACCTTCACCGC
    CACCGAGCTGATCATGTTCTACATCTTCTTCGAGACCACCCTGATCCCCACCCTGGCCATCATCACCC
    GCTGGGGCAACCAGCCCGAGCGCCTGAACGCCGGCACCTACTTCCTGTTCTACACCCTGGTGGGCA
    GCCTGCCCCTGCTGATCGCCCTGATCTACACCCACAACACCCTGGGCAGCCTGAACATCCTGCTGCT
    GACCCTGACCGCCCAGGAGCTGAGCAACAGCTGGGCCAACAACCTGATGTGGCTGGCCTACACCAT
    GGCCTTCATGGTGAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCCAAGGCCCACGTGGAGGC
    CCCCATCGCCGGCAGCATGGTGCTGGCCGCCGTGCTGCTGAAGCTGGGCGGCTACGGCATGATGCG
    CCTGACCCTGATCCTGAACCCCCTGACCAAGCACATGGCCTACCCCTTCCTGGTGCTGAGCCTGTGG
    GGCATGATCATGACCAGCAGCATCTGCCTGCGCCAGACCGACCTGAAGAGCCTGATCGCCTACAGCA
    GCATCAGCCACATGGCCCTGGTGGTGACCGCCATCCTGATCCAGACCCCCTGGAGCTTCACCGGCG
    CCGTGATCCTGATGATCGCCCACGGCCTGACCAGCAGCCTGCTGTTCTGCCTGGCCAACAGCAACTA
    CGAGCGCACCCACAGCCGCATCATGATCCTGAGCCAGGGCCTGCAGACCCTGCTGCCCCTGATGGC
    CTTCTGGTGGCTGCTTGGCCAGCCTGGCCAACCTGGCCCTGCCCCCCACCATCAACCTGCTGGGCGA
    GCTGAGCGTGCTGGTGACCACCTTCAGCTGGAGCAACATCACCCTGCTGCTGACCGGCCTGAACATG
    CTGGTGACCGCCCTGTACAGCCTGTACATGTTCACCACCACCCAGTGGGGCAGCCTGACCCACCACA
    TCAACAACATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCACCTGAGCCCCATCCTG
    CTGCTGAGCCTGAACCCCGACATCATCACCGGCTTCAGCAGCTAA
    9 ND6 ATGATGTATGCTTTGTTTCTGTTGAGTGTGGGTTTAGTAATGGGGTTTGTGGGGTTTTCTTCTAAGCCT
    TCTCCTATTTATGGGGGTTTAGTATTGATTGTTAGCGGTGTGGTCGGGTGTGTTATTATTCTGAATTTTG
    GGGGAGGTTATATGGGTTTAATGGTTTTTTTAATTTATTTAGGGGGAATGATGGTTGTCTTTGGATATAC
    TACAGCGATGGCTATTGAGGAGTATCCTGAGGCATGGGGGTCAGGGGTTGAGGTCTTGGTGAGTGTT
    TTAGTGGGGTTAGCGATGGAGGTAGGATTGGTGCTGTGGGTGAAAGAGTATGATGGGGTGGTGGTTG
    TGGTAAACTTTAATAGTGTAGGAAGCTGGATGATTTATGAAGGAGAGGGGTCAGGGTTGATTCGGGAG
    GATCCTATTGGTGCGGGGGCTTTGTATGATTATGGGCGTTTGGTTAGTAGTAGTTACTGGTTGGACATT
    GTTTGTTGGTGTATATATTGTAATTGAGATTGCTCGGGGGAATTAG
    10 opt_ND6 ATGATGTACGCCCTGTTCCTGCTGAGCGTGGGCCTGGTGATGGGCTTCGTGGGCTTCAGCAGCAAGC
    CCAGCCCCATCTACGGCGGCCTGGTGCTGATCGTGAGCGGCGTGGTGGGCTGCGTGATCATCCTGA
    ACTTCGGCGGCGGCTACATGGGCCTGATGGTGTTCCTGATCTACCTGGGCGGCATGATGGTGGTGTT
    CGGCTACACCACCGCCATGGCCATCGAGGAGTACCCCGAGGCCTGGGGCAGCGGCGTGGAGGTGC
    TGGTGAGCGTGCTGGTGGGCCTGGCCATGGAGGTGGGCCTGGTGCTGTGGGTGAAGGAGTACGACG
    GCGTGGTGGTGGTGGTGAACTTCAACAGCGTGGGCAGCTGGATGATCTACGAGGGCGAGGGCAGCG
    GCCTGATCCGCGAGGACCCCATCGGCGCCGGCGCCCTGTACGACTACGGCCGCTGGCTGGTGGTG
    GTGACCGGCTGGACCCTGTTCGTGGGCGTGTACATCGTGATCGAGATCGCCCGCGGCAACTAA
    11 ND1 ATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCATTCCTAATGCTTACCGAACGA
    AAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGCCCCTACGGGCTACTACAACC
    CTTCGCTGACGCCATAAAACTCTTCACCAAAGAGCCCCTAAAACCCGCCACATCTACCATCACCCTCT
    ACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGACCCCCCTCCCCATGCCCAAC
    CCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTAGCCTAGCCGTTTACTCAATC
    CTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCACTGCGAGCAGTAGCCCAAA
    CAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTAATGAGTGGCTCCTTTAACCT
    CTCCACCCTTATCACTAACACAAGAACACCTCTGGTTACTCCTGCCATDATGGCCCTTGGCCATGATGT
    GGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCGAAGGGGAGTCCGAACT
    AGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTCTTCATGGCCGAATACACAA
    ACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAACATATGACGCACTCTCCCCTG
    AACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCTGTTCTTATGGATTCGAACAGC
    ATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAACTTCCTACCACTCACCCTAG
    CATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCCCCTCAAACCTAA
    12 opt_ND1 ATGGCCAACCTGCTGCTGCTGATCGTGCCCATCCTGATCGCCATGGCCTTCCTGATGCTGACCGAGC
    GCAAGATCCTGGGCTACATGCAGCTGCGCAAGGGCCCCAACGTGGTGGGCCCCTACGGCCTGCTGC
    AGCCCTTCGCCGACGCCATCAAGCTGTTCACCAAGGAGCCCCTGAAGCCCGCCACCAGCACCATCAC
    CCTGTACATCACCGCCCCCACCCTGGCCCTGACCATCGCCCTGCTGCTGTGGACCCCCCTGCCCATG
    CCCAACCCCCTGGTGAACCTGAACCTGGGCCTGCTGTTCATCCTGGCCACCAGCAGCCTGGCCGTGT
    ACAGCATCCTGTGGAGCGGCTGGGCCAGCAACAGCAACTACGCCCTGATCGGCGCCCTGCGCGCCG
    TGGCCCAGACCATCAGCTACGAGGTGACCCTGGCCATCATCCTGCTGAGCACCCTGCTGATGAGCGG
    CAGCTTCAACCTGAGCACCCTGATCACCACCCAGGAGCACCTGTGGCTGCTGCTGCCCAGCTGGCCC
    CTGGCCATGATGTGGTTCATCAGCACCCTGGCCGAGACCAACCGCACCCCCTTCGACCTGGCCGAGG
    GCGAGAGCGAGCTGGTGAGCGGCTTCAACATCGAGTACGCCGCCGGCCCCTTCGCCCTGTTCTTCAT
    GGCCGAGTACACCAACATCATCATGATGAACACCCTGACCACCACCATCTTCCTGGGCACCACCTACG
    ACGCCCTGAGCCCCGAGCTGTACACCACCTACTTCGTGACCAAGACCCTGCTGCTGACCAGCCTGTT
    CCTGTGGATCCGCACCGCCTACCCCCGCTTCCGCTACGACCAGCTGATGCACCTGCTGTGGAAGAAC
    TTCCTGCCCCTGACCCTGGCCCTGCTGATGTGGTACGTGAGCATGCCCATCACCATCAGCAGCATCC
    CCCCCCAGACCTAA
    13 3′UTR GAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAA
    CACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGAC
    AGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAAT
    ACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCT
    GTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACAC
    CACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCT
    GCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAG
    CCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAAT
    AGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCT
    GGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCAC
    AGGCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCT
    GGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGG
    GTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAG
    GGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATC
    CTGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTT
    CTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTT
    TTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGA
    TGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAG
    TTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCAC
    TGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAG
    GAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    14 3′UTR* GAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAA
    CACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGAC
    AGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAAT
    ACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCT
    GTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACAC
    CACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCT
    GCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAG
    CCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAAT
    AGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCT
    GGACTGCCA
    15 COX10-ND4-3′UTR ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAGAACTATGCTAAAACTAATCGTCCCAACAATTATGTTACTACCACTGACATGGCTTTC
    CAAAAAACACATGATTTGGATCAACACAACCACCCACAGCCTAATTATTAGCATCATCCCTCTACTATTT
    TTTAACCAAATCAACAACAACCTATTTAGCTGTTCCCCAACCTTTTCCTCCGACCCCCTAACAACCCCC
    CTCCTAATGCTAACTACCTGGCTCCTACCCCTCACAATCATGGCAAGCCAACGCCACTTATCCAGTGA
    ACCACTATCACGAAAAAAACTCTACCTCTCTATGCTAATCTCCCTACAAATCTCCTTAATTATGACATTC
    ACAGCCACAGAACTAATCATGTTTTATATCTTCTTCGAAACCACACTTATCCCCACCTTGGCTATCATCA
    CCCGATGGGGCAAACCAGCCAGAACGCCTGAACGCAGGCACATACTTCCTATTCTACACCCTAGTAGG
    CTCCCTTCCCCTACTCATCGCACTAATTTACACTCACAACACCCTAGGCTCACTAAACATTCTACTACT
    CACTCTCACTGCCCAAGAACTATCAAACTCCTGGGCCAACAACTTAATGTGGCTAGCTTACACAATGG
    CTTTTATGGTAAAGATGCCTCTTTACGGACTCCACTTATGGCTCCCTAAAGCCCATGTCGAAGCCCCCA
    TCGCTGGGTCAATGGTACTTGCCGCAGTACTCTTAAAACTAGGCGGCTATGGTATGATGCGCCTCACA
    CTCATTCTCAACCCCCTGACAAAACACATGGCCTACCCCTTCCTTGTACTATCCCTATGGGGCATGATT
    ATGACAAGCTCCATCTGCCTACGACAAACAGACCTAAAATCGCTCATTGCATACTCTTCAATCAGCCAC
    ATGGCCCTCGTAGTAACAGCCATTCTCATCCAAACCCCCTGGAGCTTCACCGGCGCAGTCATTCTCAT
    GATCGCCCACGGGCTTACATCCTCATTACTATTCTGCCTAGCAAACTCAAACTACGAACGCACTCACA
    GTCGCATCATGATCCTCTCTCAAGGACTTCAAACTCTACTCCCACTAATGGCTTTTTGGTGGCTTCTAG
    CAAGCCTCGCTAACCTCGCCTTACCCCCCACTATTAACCTACTGGGAGAACTCTCTGTGCTAGTAACC
    ACGTTCTCCTGGTCAAATATCACTCTCCTACTTACAGGACTCAACATGCTAGTCACAGCCCTATACTCC
    CTCTACATGTTTACCACAACACAATGGGGCTCACTCACCCACCACATTAACAACATGAAACCCTCATTC
    ACACGAGAAAACACCCTCATGTTCATGCACCTATCCCCCATTCTCCTCCTATCCCTCAACCCCGACATC
    ATTACCGGGTTTTCCTCTTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAG
    CATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTC
    GGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAA
    TGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTC
    CTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTT
    GGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCA
    GAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGC
    ACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCAT
    AGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTT
    TGCCTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATT
    TCAGAACTCCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTT
    CCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTAC
    CTCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGA
    GAAGGGAAGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTG
    GGTGAAAAATACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTT
    TAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCA
    AATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAA
    GGTCTTGAAGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTC
    GATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGT
    AGGTAGATAACATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTT
    ACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGT
    AGAAGCTTT
    16 COX10-ND4-3′UTR* ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAGAACTATGCTAAAACTAATCGTCCCCAATTATGTTACTACCACTGACATGGCTTTC
    CAAAAAACACATGATTTGGATCAACACAACCACCCACAGCCTAATTATTAGCATCATCCCTCTACTATTT
    TTTAACCAAATCAACAACAACCTATTTAGCTGTTCCCCAACCTTTTCCTCCGACCCCCTAACAACCCCC
    CTCCTAATGCTAACTACCTGGCTCCTACCCCTCACAATCATGGCAAGCCAACGCCACTTATCCAGTGA
    ACCACTATCACGAAAAAAACTCTACCTCTCTATGCTAATCTCCCTACAAATCTCCTTAATTATGACATTC
    ACAGCCACAGAACTAATCATGTTTTATATCTTCTTCGAAACCACACTTATCCCCACCTTGGCTATCATCA
    CCCGATGGGGCAACCAGCCAGAACGCCTGAACGCAGGCACATACTTCCTATTCTACACCCTAGTAGG
    CTCCCTTCCCCTACTCATCGCACTAATTTACACTCACAACACCCTAGGCTCACTAAACATTCTACTACT
    CACTCTCACTGCCCAAGAACTATCAAACTCCTGGGCCAACAACTTAATGTGGCTAGCTTACACAATGG
    CTTTTATGGTAAAGATGCCTCTTTACGGACTCCACTTATGGCTCCCTAAAGCCCATGTCGAAGCCCCCA
    TCGCTGGGTCAATGGTACTTGCCGCAGTACTCTTAAAACTAGGCGGCTATGGTATGATGCGCCTCACA
    CTCATTCTCAACCCCCTGACAAAACACATGGCCTACCCCTTCCTTGTACTATCCCTATGGGGCATGATT
    ATGACAAGCTCCATCTGCCTACGACAAAACAGACCTAAAUCGCTCATTGCATACTCTTCAATCAGCCAC
    ATGGCCCTCGTAGTAACAGCCATTCTCATCCAAACCCCCTGGAGCTTCACCGGCGCAGTCATTCTCAT
    GATCGCCCACGGGCTTACATCCTCATTACTATTCTGCCTAGCAAACTCAAACTACGAACGCACTCACA
    GTCGCATCATGATCCTCTCTCAAGGACTTCAAACTCTACTCCCACTAATGGCTTTTTGGTGGCTTCTAG
    CAAGCCTCGCTAACCTCGCCTTACCCCCCACTATTAACCTACTGGGAGAACTCTCTGTGCTAGTAACC
    ACGTTCTCCTGGTCAAATATCACTCTCCTACTTACAGGACTCAACATGCTAGTCACAGCCCTATACTCC
    CTCTACATGTTTACCACAACACAATGGGGCTCACTCACCCACCACATTAACAACATGAAACCCTCATTC
    ACACGAGAAAACACCCTCATGTTCATGCACCTATCCCCCATTCTCCTCCTATCCCTCAACCCCGACATC
    ATTACCGGGTTTTCCTCTTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAG
    CATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTC
    GGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAA
    TGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTC
    CTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTT
    GGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCA
    GAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGC
    ACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCAT
    AGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTT
    TGCCTTGGGAGTCTCAAGCTGGACTGCCA
    17 COX10-opt_ND4-3′UTR ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAGCTATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCTCTGACCTGGCTG
    AGCAAGAAACACATGATCTGGATCAACACCACCACGCACAGCCTGATCATCAGCATCATCCCTCTGCT
    GTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCTCTGACAA
    CACCTCTGCTGATGCTGACCACCTGGCTGCTGCCCCTCACAATCATGGCCTCTCAGAGACACCTGAG
    CAGCGAGCCCCTGAGCCGGAAGAAACTGTACCTGAGCATGCTGATCTCCCTGCAGATCTCTCTGATC
    ATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTTTTCGAGACAACGCTGATCCCCACACT
    GGCCATCATCACCAGATGGGGCAACCAGCCTGAGAGACTGAACGCCGGCACCTACTTTCTGTTCTAC
    ACCCTCGTGGGCAGCCTGCCACTGCTGATTGCCCTGATCTACACCCACAACACCCTGGGCTCCCTGA
    ACATCCTGCTGCTGACACTGACAGCCCAAGAGCTGAGCAACAGCTGGGCCAACAATCTGATGTGGCT
    GGCCTACACAATGGCCTTCATGGTCAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCTAAAGCT
    CATGTGGAAGCCCCTATCGCCGGCTCTATGGTGCTGGCTGCAGTGCTGCTGAAACTCGGCGGCTACG
    GCATGATGCGGCTGACCCTGATTCTGAATCCCCTGACCAAGCACATGGCCTATCCATTTCTGGTGCTG
    AGCCTGTGGGGCATGATTATGACCAGCAGCATCTGCCTGCGGCAGACCGATCTGAAGTCCCTGATCG
    CCTACAGCTCCATCAGCCACATGGCCCTGGTGGTCACCGCCATCCTGATTCAGACCCCTTGGAGCTTT
    ACAGGCGCCGTGATCCTGATGATTGCCCACGGCCTGACAAGCAGCCTGCTGTTTTGTCTGGCCAACA
    GCAACTACGAGCGGACCCACAGCAGAATCATGATCCTGTCTCAGGGCCTGCAGACCCTCCTGCCTCT
    TATGGCTTTTTGGTGGCTGCTGGCCTCTCTGGCCAATCTGGCACTGCCTCCTACCATCAAlCTGGTGG
    GCGAGCTGAGCGTGCTGGTCACCACATTCAGCTGGTCCAATATCACCCTGCTGCTCACCGGCCTGAA
    CATGCTGGTTACAGCCCTGTACTCCCTGTACATGTTCACCACCACACAGTGGGGAAGCCTGACACACC
    ACATCAACAATATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCATCTGAGCCCCATT
    CTGCTGCTGFCCCTGAATCCTGATATCATCACCGGCTTCTCCAGCTGAGAGCACTGGGACGCCCACC
    GCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTG
    GGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATAT
    TACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTT
    CCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGG
    GGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATG
    CCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGA
    GCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGG
    TTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTG
    TGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGGTGGACTGCCAGCCCCTGTCC
    TCCCTTCACCCCCATTGCGTATGAGCATTTCAGCTCCAAGGAGTCACAGGCATCTTTATAGTTCACG
    TTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGGAT
    ACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTACA
    CAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTGTGGTGGGCTAACCAG
    CCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGATATCTCCTGAATTCAG
    AAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTTAC
    CTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCA
    CGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTTTCGATTACTCAGTCT
    CCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACAGA
    GTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGCACTTATCTGAAAT
    CTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAGCTT
    CTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    18 COX10-opt_ND4-3′UTR ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAAACTATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCTCTGACCTGGCTG
    AGCAAGAAACACATGATCTGGATCAACACCACCACGCACAGCCTGATCATCAGCATCATCCCTCTGCT
    GTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCTCTGACAA
    CACCTCTGCTGATGCTGACCACCTGGCTGCTGCCCCTCACAATCATGGCCTCTCAGAGACACCTGAG
    CAGCGAGCCCCTGAGCCGGAAGAAACTGTACCTGAGCATGCTGATCTCCCTGCAGATCTCTCTGATC
    ATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTTTTCGAGACAACGCTGATCCCCACACT
    GGCCATCATCACCAGATGGGGCAACCAGCCTGAGAGACTGAACGCCGGCACCTACTTTCTGTTCTAC
    ACCCTCGTGGGCAGCCTGCCACTGCTGATTGCCCTGATCTACACCCACAACACCCTGGGCTCCCTGA
    ACATCCTGCTGCTGACACTGACAGCCCAAGAGCTGAGCAACAGCTGGGCCAACAATCTGATGTGGCT
    GGCCTACACAATGGCCTTCATGGTCAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCTAAAGCT
    CATGTGGAAGCCCCTATCGCCGGCTCTATGGTGCTGGCTGCAGTGCTGCTGAAACTCGGCGGCTACG
    GCATGATGCGGCTGACCCTGATTCTGAATCCCCTGACCAAGCACATGGCCTATCCATTTCTGGTGCTG
    AGCCTGTGGGGCATGATTATGACCAGCAGCATCTGCCTGCGGCAGACCGATCTGAAGTCCCTGATCG
    CCTACAGCTCCATCAGCCACATGGCCCTGGTGGTCACCGCCATCCTGATTCAGACCCCTTGGAGCTTT
    ACAGGCGCCGTGATCCTGATGATTGCCCACGGCCTGACAAGCAGCCTGCTGTTTTGTCTGGCCAACA
    GCAACTACGAGCGGACCCACAGCAGAATCATGATCCTGTCTCAGGGCCTGCAGACCCTCCTGCCTCT
    TATGGCTTTTTGGTGGCTGCTGGCCTCTCTGGCCAATCTGGCACTGCCTCCTACCATCAATCTGCTGG
    GCGAGCTGAGCGTGCTGGTCACCACATTCAGCTGGTCCAATATCACCCTGCTGCTCACCGGCCTGAA
    CATGCTGGTTACAGCCCTGTACTCCCTGTACATGTTCACCACCACACAGTGGGGAAGCCTGACACACC
    ACATCAACAATATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCATCTGAGCCCCATT
    CTGCTGCTGTCCCTGAATCCTGATATCATCACCGGCTTCTCCAGCTGAGAGCACTGGGACGCCCACC
    GCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTG
    GGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATAT
    TACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTT
    CCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGG
    GGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATG
    CCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGA
    GCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGG
    TTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTG
    TGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    19 COX10-opt_ND4*-3′UTR ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAAACTATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCCCTGACCTGGCTG
    AGCAAGAAGCACATGATCTGGATCAACACCACCACCCACAGCCTGATCATCAGCATCATCCCCCTGCT
    GTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCCCTGACCA
    CCCCCCTGCTGATGCTGACCACCTGGCTGCTGCCCCTGACCATCATGGCCAGCCAGCGCCACCTGAG
    CAGCGAGCCCCTGAGCCGCAAGAAGCTGTACCTGAGCATGCTGATCAGCCTGCAGATCAGCCTGATC
    ATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTCTTCGAGACCACCCTGATCCCCACCCT
    GGCCATCATCACCCGCTGGGGCAACCAGCCCGAGCGCCTGAACGCCGGCACCTACTTCCTGTTCTAC
    ACCCTGGTGGGCAGCCTGCCCCTGCTGATCGCCCTGATCTACACCCACAACACCCTGGGCAGCCTGA
    ACATCCTGCTGCTGACCCTGACCGCCCAGGAGCTGAGCAACAGCTGGGCCAACAACCTGATGTGGCT
    GGCCTACACCATGGCCTTCATGGTGAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCCAAGGCC
    CACGTGGAGGCCCCCATCGCCGGCAGCATGGTGCTGGCCGCCGTGCTGCTGAAGCTGGGCGGCTAC
    GGCATGATGCGCCTGACCCTGATCCTGAACCCCCTGACCAAGCACATGGCCTACCCCTTCCTGGTGC
    TGAGCCTGTGGGGCATGATCATGACCAGCAGCATCTGCCTGCGCCAGACCGACCTGAAGAGCCTGAT
    CGCCTACAGCAGCATCAGCCACATGGCCCTGGTGGTGACCGCCATCCTGATCCAGACCCCCTGGAGC
    TTCACCGGCGCCGTGATCCTGATGATCGCCCACGGCCTGACCAGCAGCCTGCTGTTCTGCCTGGCCA
    ACAGCAACTACGAGCGCACCCACAGCCGCATCATGATCCTGAGCCAGGGCCTGCAGACCCTGCTGCC
    CCTGATGGCCTTCTGGTGGCTGCTGGCCAGCCTGGCCAACCTGGCCCTGCCCCCCACCATCAACCTG
    CTGGGCGAGCTGAGCGTGCTGGTGACCACCTTCAGCTGGAGCAACATCACCCTGCTGCTGACCGGC
    CTGAACATGCTGGTGACCGCCCTGTACAGCCTGTACATGTTCACCACCACCCAGTGGGGCAGCCTGA
    CCCACCACATCAACAACATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCACCTGAGC
    CCCATCCTGCTGCTGAGCCTGAACCCCGACATCATCACCGGCTTCAGCAGCTAAGAGCACTGGGACG
    CCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAAGAAGAGAA
    ATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTT
    TTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAA
    TTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCT
    CACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACT
    CCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGT
    TCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCA
    TTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCG
    GCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGGTGGACTGCCAGCC
    CCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGCATCTTTATA
    GTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCA
    GCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCCACTCCT
    CTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTGTGCTGGG
    CTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGATATCTCCT
    GAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTG
    CAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGT
    GCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTTTCGATTAC
    TCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACAACATTT
    AAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGCACTTAT
    CTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTCTGGAA
    AAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    20 COX10-opt_ND4*-3′UTR* ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAGAACTATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCCCTGACCTGGCTG
    AGCAAGAAGCACATGATCTGGATCAACACCACCACCCACAGCCTGATCATCAGCATCATCCCCCTGCT
    GTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCCCTGACCA
    CCCCCCTGCTGATGCTGACCACCTGGCTGCTGCCCCTGACCATCATGGCCAGCCAGCGCCACCTGAG
    CAGCGAGCCCCTGAGCCGCAAGAAGCTGTACCTGAGCATGCTGATCAGCCTGCAGATCAGCCTGATC
    ATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTCTTCGAGACCACCCTGATCCCCACCCT
    GGCCATCATCACCCGCTGGGGCAACCAGCCCGAGCGCCTGAACGCCGGCACCTACTTCCTGTTCTAC
    ACCCTGGTGGGCAGCCTGCCCCTGCTGATCGCCCTGATCTACACCCACAACACCCTGGGCAGCCTGA
    ACATCCTGCTGCTGACCCTGACCGCCCAGGAGCTGAGCAACAGCTGGGCCAACAACCTGATGTGGCT
    GGCCTACACCATGGCCTTCATGGTGAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCCAAGGCC
    CACGTGGAGGCCCCCATCGCCGGCAGCATGGTGCTGGCCGCCGTGCTGCTGAAGCTGGGCGGCTAC
    GGCATGATGCGCCTGACCCTGATCCTGAACCCCCTGACCAAGCACATGGCCTACCCCTTCCTGGTGC
    TGAGCCTGTGGGGCATGATCATGACCAGCAGCATCTGCCTGCGCCAGACCGACCTGAAGAGCCTGAT
    CGCCTACAGCAGCATCAGCCACATGGCCCTGGTGGTGACCGCCATCCTGATCCAGACCCCCTGGAGC
    TTCACCGGCGCCGTGATCCTGATGATCGCCCACGGCCTGACCAGCAGCCTGCTGTTCTGCCTGGCCA
    ACAGCAACTACGAGCGCACCCACAGCCGCATCATGATCCTGAGCCAGGGCCTGCAGACCCTGCTGCC
    CCTGATGGCCTTCTGGTGGCTGCTGGCCAGCCTGGCCAACCTGGCCCTGCCCCCCACCATCAACCTG
    CTGGGCGAGCTGAGCGTGCTGGTGACCACCTTCAGCTGGAGCAACATCACCCTGCTGCTGACCGGC
    CTGAACATGCTGGTGACCGCCCTGTACAGCCTGTACATGTTCACCACCACCCAGTGGGGCAGCCTGA
    CCCACCACATCAACAACATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCACCTGAGC
    CCCATCCTGCTGCTGAGCCTGAACCCCGACATCATCACCGGCTTCAGCAGCTAAGAGCACTGGGACG
    CCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAAGAAGAGAA
    ATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTT
    TTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAA
    TTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCT
    CACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACT
    CCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGT
    TCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCA
    TTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCG
    GCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    21 COX10-ND6-3′UTR ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAGAACTATGATGTATGCTTTGTTTCTGTTGAGTGTGGGTTTAGTAATGGGGTTTGTGG
    GGTTTTCTTCTAAGCCTTCTCCTATTTATGGGGGTTTAGTATTGATTGTTAGCGGTGTGGTCGGGTGTG
    TTATTATTCTGAATTTTGGGGGAGGTTATATGGGTTTAATGGTTTTTTTAATTTATTTAGGGGGAATGAT
    GGTTGTCTTTGGATATACTACAGCGATGGCTATTGAGGAGTATCCTGAGGCATGGGGGTCAGGGGTT
    GAGGTCTTGGTGAGTGTTTTAGTGGGGTTAGCGATGGAGGTAGGATTGGTGCTGTGGGTGAAAGAGT
    ATGATGGGGTGGTGGTTGTGGTAAACTTTAATAGTGTAGGAAGCTGGATGATTTATGAAGGAGAGGGG
    TCAGGGTTGATTCGGGAGGATCCTATTGGTGCGGGGGCTTTGTATGATTATGGGCGTTGGTTAGTAGT
    AGTTACTGGTTGGACATTGTTTGTTGGTGTATATATTGTAATTGAGATTGCTCGGGGGAATTAGGAGCA
    CTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAA
    GAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTT
    TTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAA
    AAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTT
    CTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACA
    CGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTG
    TCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAG
    GGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCT
    AGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGAC
    TGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGC
    ATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGAC
    TTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCG
    CCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTG
    TGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGA
    TATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGG
    GAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAG
    TCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTT
    TCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAA
    CAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTT
    GCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATG
    TCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    22 COX10-ND6-3′UTR* ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAGAACTATGATGTATGCTTTGTTTCTGTTGAGTGTGGGTTTAGTAATGGGGTTTGTGG
    GGTTTTCTTCTAAGCCTTCTCCTATTTATGGGGGTTTAGTATTGATTGTTAGCGGTGTGGTCGGGTGTG
    TTATTATTCTGAATTTTGGGGGAGGTTATATGGGTTTAATGGTTTTTTTAATTTATTTAGGGGGAATGAT
    GGTTGTCTTTGGATATACTACAGCGATGGCTATTGAGGAGTATCCTGAGGCATGGGGGTCAGGGGTT
    GAGGTCTTGGTGAGTGTTTTAGTGGGGTTAGCGATGGAGGTAGGATTGGTGCTGTGGGTGAAAGAGT
    ATGATGGGGTGGTGGTTGTGGTAAACTTTAATAGTGTAGGAAGCTGGATGATTTATGAAGGAGAGGGG
    TCAGGGTTGATTCGGGAGGATCCTATTGGTGCGGGGGCTTTGTATGATTATGGGCGTTGGTTAGTAGT
    AGTTACTGGTTGGACATTGTTTGTTGGTGTATATATTGTAATTGAGATTGCTCGGGGGAATTAGGAGCA
    CTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAA
    GAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTT
    TTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAA
    AAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTT
    CTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACA
    CGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTG
    TCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTCTGACTGAGCCAG
    GGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCT
    AGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGAC
    TGCCA
    23 COX10-opt_ND6-3′UTR ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAGAACTATGATGTACGCCCTGTTCCTGCTGAGCGTGGGCCTGGTGATGGGCTTCGTG
    GGCTTCAGCAGCAAGCCCAGCCCCATCTACGGCGGCCTGGTGCTGATCGTGAGCGGCGTGGTGGGC
    TGCGTGATCATCCTGAACTTCGGCGGCGGCTACATGGGCCTGATGGTGTTCCTGATCTACCTGGGCG
    GCATGATGGTGGTGTTCGGCTACACCACCGCCATGGCCATCGAGGAGTACCCCGAGGCCTGGGGCA
    GCGGCGTGGAGGTGCTGGTGAGCGTGCTGGTGGGCCTGGCCATGGAGGTGGGCCTGGTGCTGTGG
    GTGAAGGAGTACGACGGCGTGGTGGTGGTGGTGAACTTCAACAGCGTGGGCAGCTGGATGATCTAC
    GAGGGCGAGGGCAGCGGCCTGATCCGCGAGGACCCCATCGGCGCCGGCGCCCTGTACGACTACGG
    CCGCTGGCTGGTGGTGGTGACCGGCTGGACCCTGTTCGTGGGCGTGTACATCGTGATCGAGATCGC
    CCGCGGCAACTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTG
    TGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTC
    AGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAG
    CTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACC
    CCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCAT
    CCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGT
    GAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTT
    CCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTC
    TAACAATACCAAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGG
    GAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACT
    CCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAA
    AAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGA
    GTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGA
    AGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAA
    AATACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGC
    CAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGG
    TTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGA
    AGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTC
    GGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGAT
    AACATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGA
    GAACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTT
    T
    24 COX10-opt_ND6-3′UTR* ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAGAACTATGATGTACGCCCTGTTCCTGCTGAGCGTGGGCCTGGTGATGGGCTTCGTG
    GGCTTCAGCAGCAAGCCCAGCCCCATCTACGGCGGCCTGGTGCTGATCGTGAGCGGCGTGGTGGGC
    TGCGTGATCATCCTGAACTTCGGCGGCGGCTACATGGGCCTGATGGTGTTCCTGATCTACCTGGGCG
    GCATGATGGTGGTGTTCGGCTACACCACCGCCATGGCCATCGAGGAGTACCCCGAGGCCTGGGGCA
    GCGGCGTGGAGGTGCTGGTGAGCGTGCTGGTGGGCCTGGCCATGGAGGTGGGCCTGGTGCTGTGG
    GTGAAGGAGTACGACGGCGTGGTGGTGGTGGTGAACTTCAACAGCGTGGGCAGCTGGATGATCTAC
    GAGGGCGAGGGCAGCGGCCTGATCCGCGAGGACCCCATCGGCGCCGGCGCCCTGTACGACTACGG
    CCGCTGGCTGGTGGTGGTGACCGGCTGGACCCTGTTCGTGGGCGTGTACATCGTGATCGAGATCGC
    CCGCGGCAACTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTG
    TGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTC
    AGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAG
    CTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACC
    CCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCAT
    CCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGT
    GAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTT
    CCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTC
    TAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGG
    GAGTCTCAAGCTGGACTGCCA
    25 COX10-ND1-3′UTR ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAGAACTATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCATTCC
    TAATGCTTACCGAACGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGCCCC
    TACGGGCTACTACAACCCUCGCTGACGCCATAAAACTCTTCACCAAAGAGCCCCTAAAACCCGCCAC
    ATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGACCCC
    CCTCCCCATGCCCAACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTAGCC
    TAGCCGTTTACTCAATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCACTG
    CGAGCAGTAGCCCAAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTAATG
    AGTGGCTCCTTTAACCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATCATGG
    CCCTTGGCCATGATGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCGA
    AGGGGAGTCCGAACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTCTTCA
    TGCCCGAATACACAAACATTATTATGATGAACACCCTCACCACTACAATCUCCTAGGAACAACATATC
    ACGCACTCTCCCCTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCTGTTCT
    TATGGATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAACTTC
    CTACCACTCACCCTAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCCCCT
    CAAACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTA
    ATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGA
    TCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAG
    TCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACC
    CTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTAC
    CACCACACCACACGCACACTCCACATGCCCAGCAGAGTCGCACTTGGTGGCCAGAAAGTGTGAGCCT
    CATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGT
    GACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAA
    TACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTC
    TCAAGCTCGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTCCGTATGAGCATTTCAGAACTCCAAG
    GAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGG
    TAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACT
    ACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAG
    GAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACAT
    GTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGC
    AGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTG
    CAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTG
    ACAGGATGTUTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGCTA
    GGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATC
    CAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACAT
    TGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    26 COX10-ND1-3′UTR* ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAGAACTATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCATTCC
    TAATGCTTACCGAACGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGCCCC
    TACGGGCTACTACAACCCTTCGCTGACGCCATAAAACTCTTCACCAAAGAGCCCCTAAAACCCGCCAC
    ATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGACCCC
    CCTCCCCATGCCCAACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTAGCC
    TAGCCGTTTACTCAATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCACTG
    CGAGCAGTAGCCCAAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTAATG
    AGTGGCTCCTTTAAACCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATCATGG
    CCCTTGGCCATGATGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCGA
    AGGGGAGTCCGAACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTCTTCA
    TGGCCCGAATACACAAACATTATTATGATGAACACCCTCACCACTACAATCCCTAGGAACAACATATC
    ACGCACTCTCCCCTGTAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCTGTTCT
    TATGGATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAACTTC
    CTACCACTCACCCTAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCCCCT
    CAAACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTA
    ATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGA
    TCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAG
    TCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACC
    CTCTATTCTGTTTCTTCCTCCTCACATCGGGCTACACATACACAGCTTCCTCTTTTGCTTCCATCCTTAC
    CACCACACCACACGCACACTCCACATGCCCAGCAGAGTCGCACTTGGTGGCCAGAAAGTGTGAGCCT
    CATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGT
    GACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAA
    TACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTC
    TCAAGCTGGACTGCCA
    27 COX10-opt_ND1-3′UTR ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAAACTATGGCCAACCTGCTGCTGCTGATCGTGCCCATCCTGATCGCCATGGCCTTC
    CTGATGCTGACCGAGCGCAAGATCCTGGGCTACATGCAGCTGCGCAAGGGCCCCAACGTGGTGGGC
    CCCTACGGCCTGCTGCAGCCCTTCGCCGACGCCATCAAGCTGTTCACCAAGGAGCCCCTGAAGCCCG
    CCACCAGCACCATCACCCTGTACATCACCGCCCCCACCCTGGCCCTGACCATCGCCCTGCTGCTGTG
    GACCCCCCTGCCCATGCCCAACCCCCTGGTGAACCTGAACCTGGGCCTGCTGTTCATCCTGGCCACC
    AGCAGCCTGGCCGTGTACAGCATCCTGTGGAGCGGCTGGGCCAGCAACAGCAACTACGCCCTGATC
    GGCGCCCTGCGCGCCGTGGCCCAGACCATCAGCTACGAGGTGACCCTGGCCATCATCCTGCTGAGC
    ACCCTGCTGATGAGCGGCAGCTTCAACCTGAGCACCCTGATCACCACCCAGGAGCACCTGTGGCTGC
    TGCTGCCCAGCTGGCCCCTGGCCATGATGTGGTTCATCAGCACCCTGGCCGAGACCAACCGCACCCC
    CTTCGACCTGGCCGAGGGCGAGAGCGAGCTGGTGAGCGGCTTCAACATCGAGTACGCCGCCGGCCC
    CTTCGCCCTGTTCTTCATGGCCGAGTACACCAACATCATCATGATGAACACCCTGACCACCACCATCTT
    CCTGGGCACCACCTACGACGCCCTGAGCCCCGAGCTCTACACCACCTACTTCGTGACCAAGACCCTG
    CTGCTGACCAGCCTGTTCCTGTGGATCCGCACCGCCTACCCCCGCTTCCGCTACGACCAGCTGATGC
    ACCTGCTGTGGAAGAACTTCCTGCCCCTGACCCTGGCCCTGCTGATGTGGTACGTGAGCATGCCCAT
    CACCATCAGCAGCATCCCCCCCCAGACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCT
    GCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTA
    TAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCC
    CAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTT
    ATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAG
    CTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCAC
    TTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAG
    GCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACA
    TTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGA
    TTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGC
    GTATGAGCATTTCAGAACTCCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACTGT
    TGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCC
    CATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAA
    GGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATT
    CCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACATG
    TGCATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGT
    CTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCC
    ATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGT
    CCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCT
    AAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCC
    CCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCC
    TTCACATTTGTAGAAGCTTT
    28 COX10-opt_ND1-3′UTR* ATGGCCGCATCTCCGCACACTCTCTCCTCACGCCTCCTGACAGGTTGCGTAGGAGGCTCTGTCTGGT
    ATCTTGAAAGAAGAACTATGGCCAACCTGCTGCTGCTGATCGTGCCCATCCTGATCGCCATGGCCTTC
    CTGATGCTGACCGAGCGCAAGATCCTGGGCTACATGCAGCTGCGCAAGGGCCCCAACGTGGTGGGC
    CCCTACGGCCTGCTGCAGCCCTTCGCCGACGCCATCAAGCTGTTCACCAAGGAGCCCCTGAAGCCCG
    CCACCAGCACCATCACCCTGTACATCACCGCCCCCACCCTCGCCCTGACCATCGCCCTGCTGCTGTG
    GACCCCCCTGCCCATGCCCAACCCCCTGGTGAACCTGAACCTGGGCCTGCTGTTCATCCTGGCCACC
    AGCAGCCTGGCCGTGTACAGCATCCTGTGGAGCGGCTGGGCCAGCAACAGCAACTACGCCCTGATC
    GGCGCCCTGCGCGCCGTGGCCCAGACCATCAGCTACGAGGTGACCCTGGCCATCATCCTGCTGAGC
    ACCCTCCTGATGAGCGGCAGCTTCAACCTGAGCACCCTGATCACCACCCAGGAGCACCTGTGGCTGC
    TGCTGCCCAGCTGGCCCCTGGCCATGATGTGGTTCATCAGCACCCTGGCCGAGACCAACCGCACCCC
    CTTCGACCTGGCCGAGGGCGAGAGCGAGCTGGTGAGCGGCTTCAACATCGAGTACGCCGCCGGCCC
    CTTCGCCCTGTTCTTCATGGCCGAGTACACCAACATCATCATGATGAACACCCTGACCACCACCATCTT
    CCTGGGCACCACCTACGACGCCCTGAGCCCCGAGCTGTACACCACCTACTTCGTGACCAAGACCCTG
    CTGCTGACCAGCCTGTTCCTGTGGATCCGCACCGCCTACCCCCGCTTCCGCTACGACCAGCTGATGC
    ACCTGCTGTGGAAGAACTTCCTGCCCCTGACCCTGGCCCTGCTGATGTGGTACGTGAGCATGCCCAT
    CACCATCAGCAGCATCCCCCCCCAGACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCT
    GCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTA
    TAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCC
    CAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTT
    ATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAG
    CTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCAC
    TTGGTGGCCAAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAG
    GCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACA
    TTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGA
    TTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    29 opt_COX10-ND4-3′UTR ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACAATGCTAAAACTAATCGTCCCAACAATTATGTTACTACCACTGACATGGCTTT
    CCAAAAAACACATGATTTGGATCAACACAACCACCCACAGCCTAATTATTAGCATCATCCCTCTACTATT
    TTTTAACCAAATCAACAACAACCTATTTAGCTGTTCCCCAACCTTTTCCTCCGACCCCCTAACAACCCC
    CCTCCTAATGCTAACTACCTGGCTCCTACCCCTCACAATCATGGCAAGCCAACGCCACTTATCCAGTG
    AACCACTATCACGAAAAAAACTCTACCTCTCTATGCTAATCTCCCTACAAATCTCCTTAATTATGACATT
    CACAGCCACAGAACTAATCATGTTTTATATCTTCTTCGAAACCACACTTATCCCCACCTTGGCTATCATC
    ACCCGATGGGGCAACCAGCCAGAACGCCTGAACGCAGGCACATACTTCCTATTCTACACCCTAGTAG
    GCTCCCTTCCCCTACTCATCGCACTAATTTACACTCACAACACCCTAGGCTCACTAAACATTCTACTAC
    TCACTCTCACTGCCCAAGAACTATCAAACTCCTGGGCCAACAACTTAATGTGGCTAGCTTACACAATGG
    CTTTTATGGTAAAGATGCCTCTTTACGGACTCCACTTATGGCTCCCTAAAGCCCATGTCGAAGCCCCCA
    TCGCTGGGTCAATGGTACTTGCCGCAGTACTCTTAAAACTAGGCGGCTATGGTATGATGCGCCTCACA
    CTCATTCTCAACCCCCTGACAAAACACATGGCCTACCCCTTCCTTGTACTATCCCTATGGGGCATGATT
    ATGACAAGCTCCATCTGCCTACGACAAACAGACCTAAAATCGCTCATTGCATACTCTTCAATCAGCCAC
    ATGGCCCTCGTAGTAACAGCCATTCTCATCCAAACCCCCTGGAGCTTCACCGGCGCAGTCATTCTCAT
    GATCGCCCACGGGCTTACATCCTCATTACTATTCTGCCTAGCAAACTCAAACTACGAACGCACTCACA
    GTCGCATCATGATCCTCTCTCAAGGACTTCAAACTCTACTCCCACTAATGGCTTTTTGGTGGCTTCTAG
    CAAGCCTCGCTAACCTCGCCTTACCCCCCACTATTAACCTACTGGGAGAACTCTCTGTGCTAGTAACC
    ACGTTCTCCTGGTCAAATATCACTCTCCTACTTACAGGACTCAACATGCTAGTCACAGCCCTATACTCC
    CTCTACATGTTTACCACAACACAATGGGGCTCACTCACCCACCACATTAACAACATGAAACCCTCATTC
    ACACGAGAAAACACCCTCATGTTCATGCACCTATCCCCCATTCTCCTCCTATCCCTCAACCCCGACATC
    ATTACCGGGTTTTCCTCTTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAG
    CATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTC
    GGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAA
    TGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTC
    CTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTT
    GGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCA
    GAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGC
    ACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCAT
    AGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTT
    TGCCTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATT
    TCAGAACTCCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTT
    CCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTAC
    TCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGA
    GAAGGGAAGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTG
    GGTGAAAAATACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTT
    TAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCA
    AATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAA
    GGTCTTGAAGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTC
    GATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGT
    AGGTAGATAACATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTT
    ACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGT
    AGAAGCTTT
    30 opt_COX10-ND4-3′UTR* ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACAATGCTAAAACTAATCGTCCCAACAATTATGTTACTACCACTGACATGGCTTT
    CCAAAAAACACATGATTTGGATCAACACCCACCCACAGCCTAATTATTAGCATCATCCCTCTACTATT
    TTTTAACCAAATCAACAACAACCTATTTAGCTGTTCCCCAACCTTTTCCTCCGACCCCCTAACAACCCC
    CCTCCTAATGCTAACTACCTGGCTCCTACCCCTCACAATCATGGCAAGCCAACGCCACTTATCCAGTG
    AACCACTATCACGAAAAAAACTCTACCTCTCTATGCTAATCTCCCTACAAATCTCCTTAATTATGACATT
    CACAGCCACAGAACTAATCATGTTTTATATCTTCTTCGAAACCACACTTATCCCCACCTTGGCTATCATC
    ACCCGATGGGGCAACCAGCCAGAACGCCTGAACGCAGGCACATACTTCCTATTCTACACCCTAGTAG
    GCTCCCTTCCCCTACTCATCGCACTAATTTACACTCACAACACCCTAGGCTCACTAAACATTCTACTAC
    TCACTCTCACTGCCCAAGAACTATCAAACTCCTGGGCCAACAACTTAATGTGGCTAGCTTACACAATGG
    CTTTTATGGTAAAGATGCCTCTTTACGGACTCCACTTATGGCTCCCTAAGCCCATGTCGAAGCCCCCA
    TCGCTGGGTCAATGGTACTTGCCGCAGTACTCTTAAAACTAGGCGGCTATGGTATGATGCGCCTCACA
    CTCATTCTCAACCCCCTGACAAAACACATGGCCTACCCCTTCCTTGTACTATCCCTATGGGGCATGATT
    ATGACAAGCTCCATCTGCCTACGACAAACAGACCTAAAATCGCTCATTGCATACTCTTCAATCAGCCAC
    ATGGCCCTCGTAGTAACAGCCATTCTCATCCAAACCCCCTGGAGCTTCACCGGCGCAGTCATTCTCAT
    GATCGCCCACGGGCTTACATCCTCATTACTATTCTGCCTAGCAAACTCAAACTACGAACGCACTCACA
    GTCGCATCATGATCCTCTCTCAAGGACTTCAAACTCTACTCCCACTAATGGCTTTTTGGTGGCTTCTAG
    CAAGCCTCGCTAACCTCGCCTTACCCCCCACTATTAACCTACTGGGAGAACTCTCTGTGCTAGTAACC
    ACGTTCTCCTGGTCAAATATCACTCTCCTACTTACAGGACTCAACATGCTAGTCACAGCCCTATACTCC
    CTCTACATGTTTACCACAACACAATGGGGCTCACTCACCCACCACATTAACAACATGAAACCCTCATTC
    ACACGAGAAAACACCCTCATGTTCATGCACCTATCCCCCATTCTCCTCCTATCCCTCAACCCCGACATC
    ATTACCGGGTTTTCCTCTTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAG
    CATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTC
    GGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAA
    TGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTC
    CTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTT
    GGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCA
    GAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGC
    ACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCAT
    AGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTT
    TGCCTTGGGAGTCTCAAGCTGGACTGCCA
    31 opt_COX10-opt_ND4-3′UTR ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACAATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCTCTGACCTGGCT
    GAGCAAGAAACACATGATCTGGATCAACACCACCACGCACAGCCTGATCATCAGCATCATCCCTCTGC
    TGTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCTCTGACA
    ACACCTCTGCTGATGCTGACCACCTGGCTGCTGCCCCTCACAATCATGGCCTCTCAGAGACACCTGA
    GCAGCGAGCCCCTGAGCCGGAAGAAACTGTACCTGAGCATGCTGATCTCCCTGCAGATCTCTCTGAT
    CATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTTTTCGAGACAACGCTGATCCCCACACT
    GGCCATCATCACCAGATGGGGCAACCAGCCTGAGAGACTGAACGCCGGCACCTACTTTCTGTTCTAC
    ACCCTCGTGGGCAGCCTGCCACTGCTGATTGCCCTGATCTACACCCACAACACCCTGGGCTCCCTGA
    ACATCCTGCTGCTGACACTGACAGCCCAAGAGCTGAGCAACAGCTGGGCCAACAATCTGATGTGGCT
    GGCCTACACAATGGCCTTCATGGTCAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCTAAAGCT
    CATGTGGAAGCCCCTATCGCCGGCTCTATGGTGCTGGCTGCAGTGCTGCTGAAACTCGGCGGCTACG
    GCATGATGCGGCTGACCCTGATTCTGAATCCCCTGACCAAGCACATGGCCTATCCATTTCTGGTGCTG
    AGCCTGTGGGGCATGATTATGACCAGCAGCATCTGCCTGCGGCAGACCGATCTGAAGTCCCTGATCG
    CCTACAGCTCCATCAGCCACATGGCCCTGGTGGTCACCGCCATCCTGATTCAGACCCCTTGGAGCTTT
    ACAGGCGCCGTGATCCTGATGATTGCCCACGGCCTGACAAGCAGCCTGCTGTTTTGTCTGGCCAACA
    GCAACTACGAGCGGACCCACAGCAGAATCATGATCCTGTCTCAGGGCCTGCAGACCCTCCTGCCTCT
    TATGGCTTTTTGGTGGCTGCTGGCCTCTCTGGCCAATCTGGCACTGCCTCCTACCATCAATCTGCTGG
    GCGAGCTGAGCGTGCTGGTCACCACATTCAGCTGGTCCAATATCACCCTGCTGCTCACCGGCCTGAA
    CATGCTGGTTACAGCCCTGTACTCCCTGTACATGTTCACCACCACACAGTGGGGAAGCCTGACACACC
    ACATCAACAATATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCATCTGAGCCCCATT
    CTGCTGCTGFCCCTGAATCCTGATATCATCACCGGCTTCTCCAGCTGAGAGCACTGGGACGCCCACC
    GCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTG
    GGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATAT
    TACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTT
    CCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGG
    GGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATG
    CCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGA
    GCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGG
    TTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTG
    TGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTCC
    TCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGCATCTTTATAGTTCACG
    TTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGGAT
    ACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTACA
    CAGCACGGCTTTTTCAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAAGGGTGTGCTGGGCTAACCAG
    CCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGATATCTCCTGAATTCAG
    AAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTTAC
    CTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCA
    CGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTTTCGATTACTCAGTCT
    CCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACAGA
    GTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGCACTTATCTGAAAT
    CTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAGCTT
    CTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    32 opt_COX10-opt_ND4-3′UTR*  ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACAATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCTCTGACCTGGCT
    GAGCAAGAAACACATGATCTGGATCAACACCACCACGCACAGCCTGATCATCAGCATCATCCCTCTGC
    TGTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCTCTGACA
    ACACCTCTGCTGATGCTGACCACCTGGCTGCTGCCCCTCACAATCATGGCCTCTCAGAGACACCTGA
    GCAGCGAGCCCCTGAGCCGGAAGAAACTGTACCTGAGCATGCTGATCTCCCTGCAGATCTCTCTGAT
    CATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTTTTCGAGACAACGCTGATCCCCACACT
    GGCCATCATCACCAGATGGGGCAACCAGCCTGAGAGACTGAACGCCGGCACCTACTTTCTGTTCTAC
    ACCCTCGTGGGCAGCCTGCCACTGCTGATTGCCCTGATCTACACCCACAACACCCTGGGCTCCCTGA
    ACATCCTGCTGCTGACACTGACAGCCCAAGAGCTGAGCAACAGGTGGGCCAACAATCTGATGTGGCT
    GGCCTACACAATGGCCTTCATGGTCAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCTAAAGCT
    CATGTGGAAGCCCCTATCGCCGGCTCTATGGTGCTGGCTGCAGTGCTGCTGAAACTCGGCGGCTACG
    GCATGATGCGGCTGACCCTGATTCTGAATCCCCTGACCAAGCACATGGCCTATCCATTTCTGGTGCTG
    AGCCTGTGGGGCATGATTATGACCAGCAGCATCTGCCTGCGGCAGACCGATCTGAAGTCCCTGATCG
    CCTACAGCTCCATCAGCCACATGGCCCTGGTGGTCACCGCCATCCTGATTCAGACCCcrTGGAGCTTT
    ACAGGCGCCGTGATCCTGATGATTGCCCACGGCCTGACAAGCAGCCTGCTGTTTTGTCTGGCCAACA
    GCAACTACGAGCGGACCCACAGCAGAATCATGATCCTGTCTCAGGGCCTGCAGACCCTCCTGCCTCT
    TATGGCTTTTTGGTGGCTGCTGGCCTCTCTGGCCAATCTGGCACTGCCTCCTACCATCAATCTGCTGG
    GCGAGCTGAGCGTGCTGGTCACCACATTCAGGTGGTCCAATATCACCCTGCTGCTCACCGGCCTGAA
    CATGCTGGTTACAGCCCTGTACTCCCTGTACATGTTCACCACCACACAGTGGGGAAGCCTGACACACC
    ACATCAACAATATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCATCTGAGCCCCATT
    CTGCTGCTGTCCCTGAATCCTGATATCATCACCGGCTTCTCCAGCTGAGAGCACTGGGACGCCCACC
    GCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTG
    GGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATAT
    TACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTT
    CCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGG
    GGGTACACATACACAGGTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATG
    CCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGA
    GCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGG
    TTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTG
    TGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    33 opt_COX10-opt_ND4*-3′UTR ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACAATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCCCTGACCTGGCT
    GAGCAAGAAGCACATGATCTGGATCAACACCACCACCCACAGCCTGATCATCAGCATCATCCCCCTGC
    TGTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCCCTGACC
    ACCCCCCTGCTGATGCTGACCACCTGGCTGCTGCCCCTGACCATCATGGCCAGCCAGCGCCACCTGA
    GCAGCGAGCCCCTGAGCCGCAAGAAGCTGTACCTGAGCATGCTGATCAGCCTGCAGATCAGCCTGAT
    CATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTCTTCGAGACCACCCTGATCCCCACCC
    TGGCCATCATCACCCGCTGGGGCAACCAGCCCGAGCGCCTGAACGCCGGCACCTACTTCCTGTTCTA
    CACCCTGGTGGGCAGCCTGCCCCTGCTGATCGCCCTGATCTACACCCACAACACCCTGGGCAGCCTG
    AACATCCTGCTGCTGACCCTGACCGCCCAGGAGCTGAGCAACAGCTGGGCCAACAACCTGATGTGGC
    TGGCCTACACCATGGCCTTCATGGTGAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCCAAGGC
    CCACGTGGAGGCCCCCATCGCCGGCAGCATGGTGCTGGCCGCCGTGCTGCTGAAGCTGGGCGGCTA
    CGGCATGATGCGCCTGACCCTGATCCTGAACCCCCTGACCAAGCACATGGCCTACCCCTTCCTGGTG
    CTGAGCCTGTGGGGCATGATCATGACCAGCAGCATCTGCCTGCGCCAGACCGACCTGAAGAGCCTGA
    TCGCCTACAGCAGCATCAGCCACATGGCCCTGGTGGTGACCGCCATCCTGATCCAGACCCCCTGGAG
    CTTCACCGGCGCCGTGATCCTGATGATCGCCCACGGCCTGACCAGCAGCCTGCTGTTCTGCCTGGCC
    AACAGCAACTACGAGCGCACCCACAGCCGCATCATGATCCTGAGCCAGGGCCTGCAGACCCTGCTGC
    CCCTGATGGCCTTCTGGTGGCTGCTGGCCAGCCTGGCCAACCTGGCCCTGCCCCCCACCATCAACCT
    GCTGGGCGAGCTGAGCGTGCTGGTGACCACCTTCAGCTGGAGCAACATCACCCTGCTGCTGACCGG
    CCTGAACATGCTGGTGACCGCCCTGTACAGCCTGTACATGTTCACCACCACCCAGTGGGGCAGCCTG
    ACCCACCACATCAACAACATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCACCTGAG
    CCCCATCCTGCTGCTGAGCCTGAACCCCGACATCATCACCGGCTTCAGCAGCTAAGAGCACTGGGAC
    GCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGA
    AATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTT
    TTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGA
    ATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCC
    TCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACT
    CCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGT
    TCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCA
    TTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCG
    GCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCAGCC
    CCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGCATCTTTATA
    GTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCA
    GCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCCACTCCT
    CTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTGTGCTGGG
    CTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGATATCTCCT
    GAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTG
    CAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGT
    GCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTTTCGATTAC
    TCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACAACATTT
    AAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGCACTTAT
    CTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTCTGGAA
    AAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    34 opt_COX10-opt_ND4*-3′UTR* ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACAATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCCCTGACCTGGCT
    GAGCAAGAAGCACATGATCTGGATCAACACCACCACCCACAGCCTGATCATCAGCATCATCCCCCTGC
    TGTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCCCTGACC
    ACCCCCCTGCTGATGCTGACCACCTGGCTGCTGCCCCTGACCATCATGGCCAGCCAGCGCCACCTGA
    GCAGCGAGCCCCTGAGCCGCAAGAAGCTGTACCTGAGCATGCTGATCAGCCTGCAGATCAGCCTGAT
    CATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTCTTCGAGACCACCCTGATCCCCACCC
    TGGCCATCATCACCCGCTGGGGCAACCAGCCCGAGCGCCTGAACGCCGGCACCTACTTCCTGTTCTA
    CACCCTGGTGGGCAGCCTGCCCCTGCTGATCGCCCTGATCTACACCCACAACACCCTGGGCAGCCTG
    AACATCCTGCTGCTGACCCTGACCGCCCAGGAGCTGAGCAACAGCTGGGCCAACAACCTGATGTGGC
    TGGCCTACACCATGGCCTTCATGGTGAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCCAAGGC
    CCACGTGGAGGCCCCCATCGCCGGCAGCATGGTGCTGGCCGCCGTGCTGCTGAAGCTGGGCGGCTA
    CGGCATGATGCGCCTGACCCTGATCCTGAACCCCCTGACCAAGCACATGGCCTACCCCTTCCTGGTG
    CTGAGCCTGTGGGGCATGATCATGACCAGCAGCATCTGCCTGCGCCAGACCGACCTGAAGAGCCTGA
    TCGCCTACAGCAGCATCAGCCACATGGCCCTGGTGGTGACCGCCATCCTGATCCAGACCCCCTGGAG
    CTTCACCGGCGCCGTGATCCTGATGATCGCCCACGGCCTGACCAGCAGCCTGCTGTTCTGCCTGGCC
    AACAGCAACTACGAGCGCACCCACAGCCGCATCATGATCCTGAGC AGGGCCTGCAGACCCTGCTGC
    CCCTGATGGCCTTCTGGTGGCTGCTGGCCAGCCTGGCCAACCTGGCCCTGCCCCCCACCATCAACCT
    GCTGGGCGAGCTGAGCGTGGTGGTGACCACCTTCAGCTGGAGCAACATCACCCTGCTGCTGACCGG
    CCTGAACATGCTGGTGACCGCCCTGTACAGCCTGTACATGTTCACCACCACCCAGTGGGGCAGCCTG
    ACCCACCACATCAACAACATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCACCTGAG
    CCCCATCCTGCTGCTGAGCCTGAACCCCGACATCATCACCGGCTTCAGCAGCTAAGAGCACTGGGAC
    GCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGA
    AATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTT
    TTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGA
    ATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCC
    TCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACT
    CCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGT
    TCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCA
    TTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCG
    GCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    35 opt_COX10-ND6-3′UTR ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACAATGATGTATGCTTTGTTTCTGTTGAGTGTGGGTTTAGTAATGGGGTTTGTG
    GGGTTTTCTTCTAAGCCTTCTCCTATTTATGGGGGTTTAGTATTGATTGTTAGCGGTGTGGTCGGGTGT
    GTTATTATTCTGAATTTTGGGGGAGGTTATATGGGTTTAATGGTTTTTTTAATTTATTTAGGGGGAATGA
    TGGTTGTCTTTGGATATACTACAGCGATGGCTATTGAGGAGTATCCTGAGGCATGGGGGTCAGGGGTT
    GAGGTCTTGGTGAGTGTTTTAGTGGGGTTAGCGATGGAGGTAGGATTGGTGCTGTGGGTGAAAGAGT
    ATGATGGGGTGGTGGTTGTGGTAAACTTTAATAGTGTAGGAAGCTGGATGATTTATGAAGGAGAGGGG
    TCAGGGTTGATTCGGGAGGATCCTATTGGTGCGGGGGCTTTGTATGATTATGGGCGTTGGTTAGTAGT
    AGTTACTGGTTGGACATTGTTTGTTGGTGTATATATTGTAATTGAGATTGCTCGGGGGAATTAGGAGCA
    CTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAA
    GAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTT
    TTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAA
    AAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTT
    CTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACA
    CGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTG
    TCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAG
    GGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCT
    AGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGAC
    TGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGC
    ATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGAC
    TTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCG
    CCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTG
    TGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGA
    TATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGG
    GAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAG
    TCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTT
    TCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAA
    CAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTT
    GCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATG
    TCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    36 opt_COX10-ND6-3′UTR* ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACAATGATGTATGCTTTGTTTCTGTTGAGTGTGGGTTTAGTAATGGGGTTTGTG
    GGGTTTTCTTCTAAGCCTTCTCCTATTTATGGGGGTTTAGTATTGATTGTTAGCGGTGTGGTCGGGTGT
    GTTATTATTCTGAATTTTGGGGGAGGTTATATGGGTTTAATGGTTTTTTTAATTTATTTAGGGGGAATGA
    TGGTTGTCTTTGGATATACTACAGCGATGGCTATTGAGGAGTATCCTGAGGCATGGGGGTCAGGGGTT
    GAGGTCTTGGTGAGTGTTTTAGTGGGGTTAGCGATGGAGGTAGGATTGGTGCTGTGGGTGAAAGAGT
    ATGATGGGGTGGTGGTTGTGGTAAACTTTAATAGTGTAGGAAGCTGGATGATTTATGAAGGAGAGGGG
    TCAGGGTTGATTCGGGAGGATCCTATTGGTGCGGGGGCTTTGTATGATTATGGGCGTTGGTTAGTAGT
    AGTTACTGGTTGGACATTGTTTGTTGGTGTATATATTGTAATTGAGATTGCTCGGGGGAATTAGGAGCA
    CTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAA
    GAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTT
    TTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAA
    AAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTT
    CTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACA
    CGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTG
    TCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAG
    GGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCT
    AGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGAC
    TGCCA
    37 opt_COX10-opt_NDS-3′UTR ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACAATGATGTACGCCCTGTTCCTGCTGAGCGTGGGCCTGGTGATGGGCTTCGT
    GGGCTTCAGCAGCAAGCCCAGCCCCATCTACGGCGGCCTGGTGCTGATCGTGAGCGGCGTGGTGGG
    CTGCGTGATCATCCTGAACTTCGGCGGCGGCTACATGGGCCTGATGGTGTTCCTGATCTACCTGGGC
    GGCATGATGGTGGTGTTCGGCTACACCACCGCCATGGCCATCGAGGAGTACCCCGAGGCCTGGGGC
    AGCGGCGTGGAGGTGCTGGTGAGCGTGCTGGTGGGCCTGGCCATGGAGGTGGGCCTGGTGCTGTG
    GGTGAAGGAGTACGACGGCGTGGTGGTGGTGGTGAACTTCAACAGCGTGGGCAGCTGGATGATCTA
    CGAGGGCGAGGGCAGCGGCCTGATCCGCGAGGACCCCATCGGCGCCGGCGCCCTGTACGACTACG
    GCCGCTGGCTGGTGGTGGTGACCGGCTGGACCCTGTTCGTGGGCGTGTACATCGTGATCGAGATCG
    CCCGCGGCAACTAAGAGCACTGGGACGCCCACCGCCCCTTTCccTCCGCTGCCAGGCGAGCATGTT
    GTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCT
    CAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCA
    GCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAAC
    CCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCA
    TCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTG
    TGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCT
    TCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTT
    CTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTG
    GGAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAAC
    TCCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAAACATATAGACACTGTTGGAAGCAGTTCCTTCTA
    AAAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGA
    GTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGA
    AGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAA
    AATACATGTCCATCCTGATATCTCCTGAAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGC
    CAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGG
    TTACCTGCAGGTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGA
    AGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTC
    GGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGAT
    AACATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGA
    GAACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTT
    T
    38 opt_COX10-opt_ND6-3′UTR* ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACAATGATGTACGCCCTGTTCCTGCTGAGCGTGGGCCTGGTGATGGGCTTCGT
    GGGCTTCAGCAGCAAGCCCAGCCCCATCTACGGCGGCCTGGTGCTGATCGTGAGCGGCGTGGTGGG
    CTGCGTGATCATCCTGAACTTCGGCGGCGGCTACATGGGCCTGATGGTGTTCCTGATCTACCTGGGC
    GGCATGATGGTGGTGTTCGGCTACACCACCGCCATGGCCATCGAGGAGTACCCCGAGGCCTGGGGC
    AGCGGCGTGGAGGTGCTGGTGAGCGTGCTGGTGGGCCTGGCCATGGAGGTGGGCCTGGTGCTGTG
    GGTGAAGGAGTACGACGGCGTGGTGGTGGTGGTGAACTTCAACAGCGTGGGCAGCTGGATGATCTA
    CGAGGGCGAGGGCAGCGGCCTGATCCGCGAGGACCCCATCGGCGCCGGCGCCCTGTACGACTACG
    GCCGCTGGCTGGTGGTGGTGACCGGCTGGACCCTGTTCGTGGGCGTGTACATCGTGATCGAGATCG
    CCCGCGGCAACTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTT
    GTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCT
    CAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAATTGCATCA
    GCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAAC
    CCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCA
    TCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTG
    TGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCT
    TCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTT
    CTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTG
    GGAGTCTCAAGCTGGACTGCCA
    39 opt_COX10-ND1-3′UTR ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACAATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCATTC
    CTAATGCTTACCGAACGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGCCC
    CTACGGGCTACTACAACCCTTCGCTGACGCCATAAACTCTTCACCAAAGAGCCCCTAAAACCCGCCA
    CATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGACCC
    CCCTCCCCATGCCCAACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTAGC
    CTAGCCGTTTACTCAATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCACT
    GCGAGCAGTAGCCCAAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTAAT
    GAGTGGCTCCTTTAACCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATCATG
    GCCCTTGGCCATGATGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCG
    AAGGGGAGTCCGAACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTCTTC
    ATGGCCGAATACACAAACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAACATAT
    GACGCACTCTCCCCTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAAACCTCCCTGTTC
    TTATGGATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAACTTC
    CTACCACTCACCCTAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCCCCT
    CAAACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTA
    ATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGA
    TCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAG
    TCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACC
    CTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTAC
    CACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCT
    CATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGT
    GACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAA
    TACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTC
    TCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAAACTCCAAG
    GAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGG
    TAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACT
    ACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAG
    GAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACAT
    GTCCATCCTGATATCTCCTGAAATTCAGAAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGC
    AGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTG
    CAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTG
    ACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTA
    GGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATC
    CAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACAT
    TGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    40 opt_COX10-ND1-3′UTR* ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACAATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCATTC
    CTAATGCTTACCGAACGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGCCC
    CTACGGGCTACTACAACCCTTCGCTGACGCCATAAAACTCTTCACCAAAGAGCCCCTAAAACCCGCCA
    CATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGClCTCACCATCGCTCTTCTACTATGGACCC
    CCCTCCCCATGCCCAACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTAGC
    CTAGCCGTTTACTCAATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCACT
    GCGAGCAGTAGCCCAAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTAAT
    GAGTGGCTCCTTTAACCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATCATG
    GCCCTTGGCCATGATGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCG
    AAGGGGAGTCCGAACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTCTTC
    ATGGCCGAATACACAAACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAACATAT
    GACGCACTCTCCCCTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCTGTTC
    TTATGGATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAACTTC
    CTACCACTCACCCTAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCCCCT
    CAAACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTA
    ATTCTGGAACACAAGAAGAGAAATTGGTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGA
    TCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAG
    TCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACC
    CTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTAC
    CACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCT
    CATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGT
    GACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAA
    TACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTC
    TCAAGCTGGACTGCCA
    41 opt_COX10-opt_ND1-3′UTR ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACAATGGCCAACCTGCTGCTGCTGATCGTGCCCATCCTGATCGCCATGGCCTT
    CCTGATGCTGACCGAGCGCAAGATCCTGGGCTACATGCAGCTGCGCAAGGGCCCCAACGTGGTGGG
    CCCCTACGGCCTGCTGCAGCCCTTCGCCGACGCCATCAAGCTGTTCACCAAGGAGCCCCTGAAGCCC
    GCCACCAGCACCATCACCCTGTACATCACCGCCCCCACCCTGGCCCTGACCATCGCCCTGCTGCTGT
    GGACCCCCCTGCCCATGCCCAACCCCCTGGTGAACCTGAACCTGGGCCTGCTGTTCATCCTGGCCAC
    CAGCAGCCTGGCCGTGTACAGCATCCTGTGGAGCGGCTGGGCCAGCAACAGCAACTACGCCCTGAT
    CGGCGCCCTGCGCGCCGTGGCCCAGACCATCAGCTACGAGGTGACCCTGGCCATCATCCTGCTGAG
    CACCCTGCTGATGAGCGGCAGCTTCAACCTGAGCACCCTGATCACCACCCAGGAGCACCTGTGGCTG
    CTGCTGCCCAGCTGGCCCCTGGCCATGATGTGGTTCATCAGCACCCTGGCCGAGACCAACCGCACCC
    CCTTCGACCTGGCCGAGGGCGAGAGCGAGCTGGTGAGCGGCTTCAACATCGAGTACGCCGCCGGCC
    CCTTCGCCCTGTTCTTCATGGCCGAGTACACCAACATCATCATGATGAACACCCTGACCACCACCATC
    TTCCTGGGCACCACCTACGACGCCCTGAGCCCCGAGCTGTACACCACCTACTTCGTGACCAAGACCC
    TGCTGCTGACCAGCCTGTTCCTGTGGATCCGCACCGCCTACCCCCGCTTCCGCTACGACCAGCTGAT
    GCACCTGCTGTGGAAGAACTTCCTGCCCCTGACCCTGGCCCTGCTGATGTGGTACGTGAGCATGCCC
    ATCACCATCAGCAGCATCCCCCCCCAGACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCG
    CTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGAT
    TATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCC
    CCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTT
    TATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACA
    GCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCA
    CTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAA
    GGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACA
    CATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGG
    GATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATT
    GCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACT
    GTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCAC
    CCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTC
    AAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACA
    TTCCTGTCCCTTGGGTGAAASATACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACA
    TGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTG
    GTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTC
    CCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTG
    GTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGT
    CTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTG
    CCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAG
    CCTTCACATTTGTAGAAGCTTT
    42 opt_COX10-opt_ND1-3′UTR ATGGCCGCCTCTCCACACACACTGAGTAGCAGACTGCTGACCGGCTGTGTTGGCGGCTCTGTGTGGT
    ATCTGGAACGGCGGACAATGGCCAACCTGCTGCTGCTGATCGTGCCCATCCTGATCGCCATGGCCTT
    CCTGATGCTGACCGAGCGCAAGATCCTGGGCTACATGCAGCTGCGCAAGGGCCCCAACGTGGTGGG
    CCCCTACGGCCTGCTGCAGCCCTTCGCCGACGCCATCAAGCTGTTCACCAAGGAGCCCCTGAAGCCC
    GCCACCAGCACCATCACCCTGTACATCACCGCCCCCACCCTGGCCCTGACCATCGCCCTGCTGCTGT
    GGACCCCCCTGCCCATGCCCAACCCCCTGGTGAACCTGAACCTGGGCCTGCTGTTCATCCTGGCCAC
    CAGCAGCCTGGCCGTGTACAGCATCCTGTGGAGCGGCTGGGCCAGCAACAGCAACTACGCCCTGAT
    CGGCGCCCTGCGCGCCGTGGCCCAGACCATCAGCTACGAGGTGACCCTGGCCATCATCCTGCTGAG
    CACCCTGCTGATGAGCGGCAGCTTCAACCTGAGCACCCTGATCACCACCCAGGAGCACCTGTGGCTG
    CTGCTGCCCAGCTGGCCCCTGGCCATGATGTGGTTCATCAGCACCCTGGCCGAGACCAACCGCACCC
    CCTTCGACCTGGCCGAGGGCGAGAGCGAGCTGGTGAGCGGCTTCAACATCGAGTACGCCGCCGGCC
    CCTTCGCCCTGTTCTTCATGGCCGAGTACACCAACATCATCATGATGAACACCCTGACCACCACCATC
    TTCCTGGGCACCACCTACGACGCCCTGAGCCCCGAGCTGTACACCACCTACTTCGTGACCAAGACCC
    TGCTGCTGACCAGCCTGTTCCTGTGGATCCGCACCGCCTACCCCCGCTTCCGCTACGACCAGCTGAT
    GCACCTGCTGTGGAAGAACTTCCTGCCCCTGACCCTGGCCCTGCTGATGTGGTACGTGAGCATGCCC
    ATCACCATCAGCAGCATCCCCCCCCAGACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCG
    CTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGAT
    TATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCC
    CCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTT
    TATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACA
    GCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCA
    CTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAA
    GGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACA
    CATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGG
    GATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    43 opt_COX10*-ND4-3′UTR ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACCATGCTAAAACTAATCGTCCCAACAATTATGTTACTACCACTGACATGGC
    TTTCCAAAAAACACATGATTTGGATCAACACAACCACCCACAGCCTAATTATTAGCATCATCCCTCTACT
    ATTTTTTAACCAAATCAACAACAACCTATTTAGCTGTTCCCCAACCTTTTCCTCCGACCCCCTAACAACC
    CCCCTCCTAATGCTAACTACCTGGCTCCTACCCCTCACAATCATGGCAAGCCAACGCCACTTATCCAG
    TGAACCACTATCACGAAAAAAACTCTACCTCTCTATGCTAATCTCCCTACAAATCTCCTTAATTATGACA
    TTCACAGCCACAAACTAATCATGTTTTATATCTTCTTCGAAACCACACTTATCCCCACCTTGGCTATCA
    TCACCCGATGGGGCAACCAGCCAGAACGCCTGAACGCAGGCACATACTTCCTATTCTACACCCTAGTA
    GGCTCCCTTCCCCTACTCATCGCACTAATTTACACTCACAACACCCTAGGCTCACTAAACATTCTACTA
    CTCACTCTCACTGCCCAAGAACTATCAAACTCCTGGGCCAACAACTTAATGTGGCTAGCTTACACAATG
    GCTTTTATGGTAAAGATGCCTCTTTACGGACTCCACTTATGGCTCCCTAAAGCCCATGTCGAAGCCCC
    CATCGCTGGGTCAATGGTACTTGCCGCAGTACTCTTAAAACTAGGCGGCTATGGTATGATGCGCCTCA
    CACTCATTCTCAACCCCCTGACAAAACACATGGCCTACCCCTTCCTTGTACTATCCCTATGGGGCATGA
    TTATGACAAGCTCCATCTGCCTACGACAAACAGACCTAAAATCGCTCATTGCATACTCTTCAATCAGCC
    ACATGGCCCTCGTAGTAACAGCCATTCTCATCCAAACCCCCTGGAGCTTCACCGGCGCAGTCATTCTC
    ATGATCGCCCACGGGCTTACATCCTCATTACTATTCTGCCTAGCAAACTCAAACTACGAACGCACTCAC
    AGTCGCATCATGATCCTCTCTCAAGGACTTCAAACTCTACTCCCACTAATGGCTTTTTGGTGGCTTCTA
    GCAAGCCTCGCTAACCTCGCCTTACCCCCCACTATTAACCTACTGGGAGAACTCTCTGTGCTAGTAAC
    CACGTTCTCCTGGTCAAATATCACTCTCCTACTTACAGGACTCAACATGCTAGTCACAGCCCTATACTC
    CCTCTACATGTTTACCACAACACAATGGGGCTCACTCACCCACCACATTAACAACATGAAACCCTCATT
    CACACGAGAAAACACCCTCATGTTCATGCACCTATCCCCCATTCTCCTCCTATCCCTCAACCCCGACAT
    CATTACCGGGTTTTCCTCTTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGA
    GCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATT
    CGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAA
    ATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCT
    CCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTT
    TGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCC
    AGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAG
    CACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACC
    ATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATG
    TTTGCCTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCA
    TTTCAGAACTCCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAG
    TTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGT
    ACCTCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATT
    GAGAAGGGAAGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCT
    TGGGTGAAAAATACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGC
    TTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTAC
    CAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCA
    AAGGTCTTGAAGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGAT
    TCGATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATT
    GTAGGTAGATAACATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTAT
    TTACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTT
    GTAGAAGCTTT
    44 opt_COX10*-ND4-3′UTR* ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACCATGCTAAAACTAATCGTCCCAACAATTATGTTACTACCACTGACATGGC
    TTTCCAAAAAACACATGATTTGGATCAACACAACCACCCACAGCCTAATTATTAGCATCATCCCTCTACT
    ATTTTTTAACCAAATCAACAACAACCTATTTAGCTGTTCCCCAACCTTTTCCTCCGACCCCCTAACAACC
    CCCCTCCTAATGCTAACTACCTGGCTCCTACCCCTCACAATCATGGCAAGCCAACGCCACTTATCCAG
    TGAACCACTATCACGAAAAAAACTCTACCTCTCTATGCTAATCTCCCTACAAATCTCCTTAATTATGACA
    TTCACAGCCACAGAACTAATCATGTTTTATATCTTCTTCGAAACCACACTTATCCCCACCTTGGCTATCA
    TCACCCGATGGGGCAACCAGCCAGAACGCCTGAACGCAGGCACATACTTCCTATTCTACACCCTAGTA
    GGCTCCCTTCCCCTACTCATCGCACTAATTTACACTCACAACACCCTAGGCTCACTAAACATTCTACTA
    CTCACTCTCACTGCCCAAGAACTATCAAACTCCTGGGCCAACAACTTAATGTGGCTAGCTTACACAATG
    GCTTTTATGGTAAAGATGCCTCTTTACGGACTCCACTTATGGCTCCCTAAAGCCCATGTCGAAGCCCC
    CATCGCTGGGTCAATGGTACTTGCCGCAGTACTCTTAAAACTAGGCGGCTATGGTATGATGCGCCTCA
    CACTCATTCTCAACCCCCTGACAAAACACATGGCCTACCCCTTCCTTGTACTATCCCTATGGGGCATGA
    TTATGACAAGCTCCATCTGCCTACGACAAACAGACCTAAAATCGCTCATTGCATACTCTTCAATCAGCC
    ACATGGCCCTCGTAGTAACAGCCATTCTCATCCAAACCCCCTGGAGCTTCACCGGCGCAGTCATTCTC
    ATGATCGCCCACGGGCTTACATCCTCATTACTATTCTGCCTAGCAAACTCAAACTACGAACGCACTCAC
    AGTCGCATCATGATCCTCTCTCAAGGACTTCAAACTCTACTCCCACTAATGGCTTTTTGGTGGCTTCTA
    GCAAGCCTCGCTAACCTCGCCTTACCCCCCACTATTbACCTACTGGGAGAACTCTCTGTGCTAGTAAC
    CACGTTCTCCTGGTCAAATATCACTCTCCTACTTACAGGACTCAACATGCTAGTCACAGCCCTATACTC
    CCTCTACATGTTTACCACAACACAATGGGGCTCACTCACCCACCACATTAACAACATGAAACCCTCATT
    CACACGAGAAAACACCCTCATGTTCATGCACCTATCCCCCATTCTCCTCCTATCCCTCAACCCCGACAT
    CATTACCGGGTTTTCCTCTTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGA
    GCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATT
    CGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAA
    ATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCT
    CCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTT
    TGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCC
    AGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAG
    CACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACC
    ATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATG
    TTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    45 opt_COX10*-opt_ND4-3′UTR ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACCATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCTCTGACCTGG
    CTGAGCAAGAAACACATGATCTGGATCAACACCACCACGCACAGCCTGATCATCAGCATCATCCCTCT
    GCTGTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCTCTGA
    CAACACCTCTGCTGATGCTGACCACCTGGCTGCTGCCCGTCACAATCATGGCCTCTCAGAGACACCTG
    AGCAGCGAGCCCGTGAGCCGGAAGAAACTGTACCTGAGCATGCTGATCTCCCTGCAGATCTCTCTGA
    TCATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTTTTCGAGACAACGCTGATCCCCACAC
    TGGCCATCATCACCAGATGGGGCAACCAGCCTGAGAGACTGAACGCCGGCACCTACTTTCTGTTCTA
    CACCCTCGTGGGCAGCCTGCCACTGCTGATTGCCCTGATCTACACCCACAACACCCTGGGCTCCCTG
    AACATCCTGCTGCTGACACTGACAGCCCAAGAGCTGAGCAACAGGTGGGCCAACAATCTGATGTGGC
    TGGCCTACACAATGGCCTTCATGGTCAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCTAAAGC
    TCATGTGGAAGCCCCTATCGCCGGCTCTATGGTGCTGGCTGCAGTGCTGCTGAACTCGGCGGCTAC
    GGCATGATGCGGCTGACCCTGATTCTGAATCCCCTGACCAAGCACATGGCCTATCCATTTCTGGTGCT
    GAGCCTGTGGGGCATGATTATGACCAGCAGCATCTGCCTGCGGCAGACCGATCTGAAGTCCCTGATC
    GCCTACAGCTCCATCAGCCACATGGCCCTGGTGGTCACCGCCATCCTGATTCAGACCCCTTGGAGCT
    TTACAGGCGCCGTGATCCTGATGATTGCCCACGGCCTGACAAGCAGCCTGCTGTTTTGTCTGGCCAA
    CAGCAACTACGAGCGGACCCACAGCAGAATCATGATCCTGTCTCAGGGCCTGCAGACCCTCCTGCCT
    CTTATGGCTTTTTGGTGGCTGCTGGCCTCTCTGGCCAATCTGGCACTGCCTCCTACCATCAATCTGCT
    GGGCGAGCTGAGCGTGCTGGTCACCACATTCAGCTGGTCCAATTATCACCCTGCTGCTCACCGGCCTG
    AACATGCTGGTTACAGCCCTGTACTCCCTGTACATGTTCACCACCACACAGTGGGGAAGCCTGACACA
    CCACATCAACAATATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCATCTGAGCCCCA
    TTCTGCTGCTGTCCCTGAATCCTGATATCATCACCGGCTTCTCCAGCTGAGAGCACTGGGACGCCCAC
    CGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCT
    GGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAAT
    ATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTT
    TCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATG
    GGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACAT
    GCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTG
    AGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTG
    GTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCT
    GTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTC
    CTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGCATCTTTATAGTTCAC
    GTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGG
    ATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTA
    CACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTGTGCTGGGCTAACC
    AGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGATATCTCCTGAATTC
    AGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTT
    ACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCC
    CACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTTTCGATTACTCAGT
    CTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACA
    GAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGCACTTATCTGA
    AATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAG
    CTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    46 opt_COX10*-opt_ND4-3′UTR* ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACCATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCTCTGACCTGG
    CTGAGCAAGAAACACATGATCTGGATCAACACCACCACGCACAGCCTGATCATCAGCATCATCCCTCT
    GCTGTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCTCTGA
    CAACACCTCTGCTGATGCTGACCACCTGGCTGCTGCCCCTCACAATCATGGCCTCTCAGAGACACCTG
    AGCAGCGAGCCCCTGAGCCGGAAGAAACTGTACCTGAGCATGCTGATCTCCCTGCAGATCTCTCTGA
    TCATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTTTTCGAGACAACGCTGATCCCCACAC
    TGGCCATCATCACCAGATGGGGCAACCAGCCTGAGAGACTGAACGCCGGCACCTACTTTCTGTTCTA
    CACCCTCGTGGGCAGCCTGCCACTGCTGATTGCCCTGATCTACACCCACAACACCCTGGGCTCCCTG
    AACATCCTGCTGCTGACACTGACAGCCCAAGAGCTGAGCAACAGCTGGGCCAACAATCTGATGTGGC
    TGGCCTACACAATGGCCTTCATGGTCAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCTAAAGC
    TCATGTGGAAGCCCCTATCGCCGGCTCTATGGTGCTGGCTGCAGTGCTGCTGAAACTCGGCGGCTAC
    GGCATGATGCGGCTGACCCTGATTCTGAATCCCCTGACCAAGCACATGGCCTATCCATTTCTGGTGCT
    GAGCCTGTGGGGCATGATTATGACCAGCAGCATCTGCCTGCGGCAGACCGATCTGAAGTCCCTGATC
    GCCTACAGCTCCATCAGCCACATGGCCCTGGTGGTCACCGCCATCCTGATTCAGACCCCTTGGAGCT
    TTACAGGCGCCGTGATCCTGATGATTGCCCACGGCCTGACAAGCAGCCTGCTGTTTTGTCTGGCCAA
    CAGCAACTACGAGCGGACCCACAGCAGAATCATGATCCTGTCTCAGGGCCTGCAGACCCTCCTGCCT
    CTTATGGCTTTTTGGTGGCTGCTGGCCTCTCTGGCCAATCTGGCACTGCCTCCTACCATCAATCTGCT
    GGGCGAGCTGAGCGTGCTGGTCACCACATTCAGCTGGTCCAATATCACCCTGCTGCTCACCGGCCTG
    AACATGCTGGTTACAGCCCTGTACTCCCTGTACATGTTCACCACCACACAGTGGGGAAGCCTGACACA
    CCACATCAACAATATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCATCTGAGCCCCA
    TTCTGCTGCTGTCCCTGAATCCTGATATCATCACCGGCTTCTCCAGCTGAGAGCACTGGGACGCCCAC
    CGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCT
    GGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAAT
    ATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTT
    TCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATG
    GGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACAT
    GCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTG
    AGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTG
    GTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCT
    GTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    47 opt_COX10*-opt_ND4*-3′UTR ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACCATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCCCTGACCTGG
    CTGAGCAAGAAGCACATGATCTGGATCAACACCACCACCCACAGCCTGATCATCAGCATCATCCCCCT
    GCTGTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCCCTGA
    CCACCCCCCTGCTGATGCTGACCACCTGGCTGCTGCCCCTGACCATCATGGCCAGCCAGCGCCACCT
    GAGCAGCGAGCCCCTGAGCCGCAAGAAGCTGTACCTGAGCATGCTGATCAGCCTGCAGATCAGCCTG
    ATCATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTCTTCGAGACCACCCTGATCCCCAC
    CCTGGCCATCATCACCCGCTGGGGCAACCAGCCCGAGCGCCTGAACGCCGGCACCTACTTCCTGTTC
    TACACCCTGGTGGGCAGCCTGCCCCTGCTGATCGCCCTGATCTACACCCACAACACCCTGGGCAGCC
    TGAACATCCTGCTGCTGACCCTGACCGCCCAGGAGCTGAGCAACAGCTGGGCCAACAACCTGATGTG
    GCTGGCCTACACCATGGCCTTCATGGTGAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCCAAG
    GCCCACGTGGAGGCCCCCATCGCCGGCAGCATGGTGCTGGCCGCCGTGCTGCTGAAGCTGGGCGG
    CTACGGCATGATGCGCCTGACCCTGATCCTGAACCCCCTGACCAAGCACATGGCCTACCCCTTCCTG
    GTGCTGAGCCTGTGGGGCATGATCATGACCAGCAGCATCTGCCTGCGCCAGACCGACCTGAAGAGC
    CTGATCGCCTACAGCAGCATCAGCCACATGGCCCTGGTGGTGACCGCCATCCTGATCCAGACCCCCT
    GGAGCTTCACCGGCGCCGTGATCCTGATGATCGCCCACGGCCTGACCAGCAGCCTGCTGTTCTGCCT
    GGCCAACAGCAACTACGAGCGCACCCACAGCCGCATCATGATCCTGAGCCAGGGCCTGCAGACCCT
    GCTGCCCCTGATGGCCTTCTGGTGGCTGCTGGCCAGCCTGGCCAACCTGGCCCTGCCCCCCACCAT
    CAACCTGCTGGGCGAGCTGAGCGTGCTGGTGACCACCTTCAGCTGGAGCAACATCACCCTGCTGCTG
    ACCGGCCTGAACATGCTGGTGACCGCCCTGTACAGCCTGTACATGTTCACCACCACCCAGTGGGGCA
    GCCTGACCCACCACATCAACAACATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCAC
    CTGAGCCCCATCCTGGTGCTGAGCCTGAACCCCGACATCATCACCGGCTTCAGCAGCTAAGAGCACT
    GGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGA
    AGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTT
    TTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAA
    AAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCT
    TCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACG
    CACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTC
    TGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGG
    CCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAG
    GACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTG
    CCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGCAT
    CTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTT
    AATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCC
    ACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTGTG
    CTGGGCTbACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGATA
    TCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGA
    ATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTC
    CTTTGTGCTCCCACGGGTCTACAGAGTCCCAlCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTTTC
    GATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACA
    ACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGC
    ACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTC
    TGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    48 opt_COX10*-opt_ND4*-3′UTR ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACCATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCCCTGACCTGG
    CTGAGCAAGAAGCACATGATCTGGATCAACACCACCACCCACAGCCTGATCATCAGCATCATCCCCCT
    GCTGTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCCCTGA
    CCACCGCCCTGCTGATGCTGACCACCTGGCTGCTGCCCCTGACCATCATGGCCAGCCAGCGCCACCT
    GAGCAGCGAGCCCCTGAGCCGCAAGAAGCTGTACCTGAGCATGCTGATCAGCCTGCAGATCAGCCTG
    ATCATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTCTTCGAGACCACCCTGATCCCCAC
    CCTGGCCATCATCACCCGCTGGGGCAACCAGCCCGAGCGCCTGAACGCCGGCACCTACTTCCTGTTC
    TACACCCTGGTGGGCAGCCTGCCCCTGCTGATCGCCCTGATCTACACCCACAACACCCTGGGCAGCC
    TGAACATCCTGCTGCTGACCCTGACCGCCCAGGAGCTGAGCAACAGCTGGGCCAACAACCTGATGTG
    GCTGGCCTACACCATGGCCTTCATGGTGAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCCAAG
    GCCCACGTGGAGGCCCCCATCGCCGGCAGCATGGTGCTGGCCGCCGTGCTGCTGAAGCTGGGCGG
    CTACGGCATGATGCGCCTGACCCTGATCCTGAACCGCCTGACCAAGCACATGGCCTACCCGTTCCTG
    GTGCTGAGCCTGTGGGGCATGATCATGACCAGCAGCATCTGCCTGCGCCAGACCGACCTGAAGAGC
    CTGATCGCCTACAGCAGCATCAGCCACATGGCCCTGGTGGTGACCGCCATCCTGATCCAGACCCCCT
    GGAGCTTCACCGGCGCCGTGATCCTGATGATCGCCCACGGCCTGACCAGCAGCCTGCTGTTCTGCCT
    GGCCAACAGCAACTACGAGCGCACCCACAGCCGCATCATGATCCTGAGCCAGGGCCTGGAGACCCT
    GCTGCCCCTGATGGCCTTCTGGTGGCTGCTGGCCAGCCTGGCCAACCTGGCCCTGCCCCCCACCAT
    CAACCTGCTGGGCGAGCTGAGCGTGCTGGTGACCACCTTCAGCTGGAGCAACATCACCCTGCTGCTG
    ACCGGCCTGAACATGCTGGTGACCGCCCTGTACAGCCTGTACATGTTCACCACCACCCAGTGGGGCA
    GCCTGACCCACCACATCAACAACATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCAC
    CTGAGCCCCATCCTGGTGCTGAGCCTGAACCCCGACATCATCACCGGCTTCAGCAGCTAAGAGCACT
    GGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGA
    AGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTT
    TTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAA
    AAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCCATAATGTTTCT
    TCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACG
    CACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTC
    TGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGG
    CCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAG
    GACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTG
    CCA
    49 opt_COX10*-ND6-3′UTR ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACCATGATGTATGCTTTGTTTCTGTTGAGTGTGGGTTTAGTAATGGGGTTTG
    TGGGGTTTTCTTCTAAGCCTTCTCCTATTTATGGGGGTTTAGTATTGATTGTTAGCGGTGTGGTCGGGT
    GTGTTATTATTCTGAATTTTGGGGGAGGTTATATGGGTTTAATGGTTTTTTTAATTTATTTAGGGGGAAT
    GATGGTTGTCTTTGGATATACTACAGCGATGGGTATTGAGGAGTATCCTGAGGCATGGGGGTCAGGG
    GTTGAGGTCTTGGTGAGTGTTTTAGTGGGGTTAGCGATGGAGGTAGGATTGGTGCTGTGGGTGAAAG
    AGTATGATGGGGTGGTGGTTGTGGTAAACTTTAATAGTGTAGGAAGCTGGATGATTTATGAAGGAGAG
    GGGTCAGGGTTGATTCGGGAGGATCCTATTGGTGCGGGGGCTTTGTATGATTATGGGCGTTGGTTAG
    TAGTAGTTACTGGTTGGACATTGTTTGTTGGTGTATATATTGTAATTGAGATTGGTCGGGGGAATTAGG
    AGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAAC
    ACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACA
    GTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATA
    CAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTG
    TTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACC
    ACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTG
    CTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCC
    AGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAG
    CTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGG
    ACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAG
    GCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGG
    ACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGT
    CGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGG
    GTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCC
    TGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCT
    GGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTT
    TAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGGTTGACAGGATG
    TTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTT
    AAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACT
    GTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGG
    AATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGGTTT
    50 opt_COX10*-ND6-3′UTR* ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACCATGATGTATGCTTTGTTTCTGTTGAGTGTGGGTTTAGTAATGGGGTTTG
    TGGGGTTTTCTTCTAAGCCTTCTCCTATTTATGGGGGTTTAGTATTGATTGTTAGCGGTGTGGTCGGGT
    GTGTTATTATTCTGAATTTTGGGGGAGGTTATATGGGTTTAATGGTTTTTTTAATTTATTTAGGGGGAAT
    GATGGTTGTCTTTGGATATACTACAGCGATGGCTATTGAGGAGTATCCTGAGGCATGGGGGTCAGGG
    GTTGAGGTCTTGGTGAGTGTTTTAGTGGGGTTAGCGATGGAGGTAGGATTGGTGGTGTGGGTGAAAG
    AGTATGATGGGGTGGTGGTTGTGGTAAACTTTAATAGTGTAGGAAGCTGGATGATTTATGAAGGAGAG
    GGGTCAGGGTTGATTCGGGAGGATCCTATTGGTGCGGGGGCTTTGTATGATTATGGGCGTTGGTTAG
    TAGTAGTTACTGGTTGGACATTGTTTGTTGGTGTATATATTGTAATTGAGATTGCTCGGGGGAATTAGG
    AGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAAC
    ACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACA
    GTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATA
    CAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTG
    TTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACC
    ACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTG
    CTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCC
    AGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAG
    CTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGG
    ACTGCCA
    51 opt_COX10*-opt_ND6-3′UTR ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACCATGATGTACGCCCTGTTCCTGCTGAGCGTGGGCCTGGTGATGGGCTTC
    GTGGGCTTCAGCAGCAAGCCCAGCCCCATCTACGGCGGCCTGGTGCTGATCGTGAGCGGCGTGGTG
    GGCTGCGTGATCATCCTGAACTTCGGCGGCGGCTACATGGGCCTGATGGTGTTCCTGATCTACCTGG
    GCGGCATGATGGTGGTGTTCGGCTACACCACCGCCATGGCCATCGAGGAGTACCCCGAGGCCTGGG
    GCAGCGGCGTGGAGGTGCTGGTGAGCGTGCTGGTGGGCCTGGCCATGGAGGTGGGCCTGGTGCTG
    TGGGTGAAGGAGTACGACGGCGTGGTGGTGGTGGTGAACTTCAACAGCGTGGGCAGCTGGATGATC
    FACGAGGGCGAGGGCAGCGGCCTGATCCGCGAGGACCCCATCGGCGCCGGCGCCCTGTACGACTA
    CGGCCGCTGGCTGGTGGTGGTGACCGGCTGGACCCTGTTCGTGGGCGTGTACATCGTGATCGAGAT
    CGCCCGCGGCAACTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATG
    TTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTG
    CTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCAT
    CAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCA
    ACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTC
    CATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAG
    TGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCC
    CTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCC
    TTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCT
    TGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGA
    ACTCCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTC
    TAAAAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTG
    GAGTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGG
    GAAGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGA
    AAAATACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGA
    GCCAGAAGCAGGGTTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATAC
    GGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAASAGGTCT
    TGAAGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTG
    GTCGGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTA
    GATAACATCCAATCACTGTTTGCACTTATCTGAAATCCTCTTGGCTGCCCCCAGGTATTTACTGT
    GGAGAACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAAG
    CTTT
    52 opt_COX10*-opt_ND6-3′UTR* ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACCATGATGTACGCCCTGTTCCTGCTGAGCGTGGGCCTGGTGATGGGCTTC
    GTGGGCTTCAGCAGCAAGCCCAGCCCCATCTACGGCGGCCTGGTGCTGATCGTGAGCGGCGTGGTG
    GGCTGCGTGATCATCCTGAACTTCGGCGGCGGCTACATGGGCCTGATGGTGTTCCTGATCTACCTGG
    GCGGCATGATGGTGGTGTTCGGCTACACCACCGCCATGGCCATCGAGGAGTACCCCGAGGCCTGGG
    GCAGCGGCGTGGAGGTGCTGGTGAGCGTGCTGGTGGGCCTGGCCATGGAGGTGGGCCTGGTGCTG
    TGGGTGAAGGAGTACGACGGCGTGGTGGTGGTGGTGAACTTCAACAGCGTGGGCAGCTGGATGATC
    TACGAGGGCGAGGGCAGCGGCCTGATCCGCGAGGACCCCATCGGCGCCGGCGCCCTGTACGACTA
    CGGCCGCTGGCTGGTGGTGGTGACCGGCTGGACCCTGTTCGTGGGCGTGTACATCGTGATCGAGAT
    CGCCCGCGGCAACTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATG
    TTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTG
    CTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCAT
    CAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCA
    ACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTC
    CATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAG
    TGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCC
    CTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCC
    TTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCT
    TGGGAGTCTCAAGCTGGACTGCCA
    53 opt_COX10*-opt_ND6-3′UTR* ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACCATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCAT
    TCCTAATGCTTACCGAACGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGC
    CCCTACGGGCTACTACAACCCTTCGCTGACGCCATAAAACTCTTCACCAAAGAGCCCCTAAAACCCGC
    CACATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGAC
    CCCCCTCCCCATGCCCAACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTA
    GCCTAGCCGTTTACTCAATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCA
    CTGCGAGCAGTAGCCCAAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTA
    ATGAGTGGCTCCTTTAACCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATCA
    TGGCCCTTGGCCATGATGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGC
    CGAAGGGGAGTCCGAACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTC
    TTCATGGCCGAATACACAAACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAACA
    TATGACGCACTCTCCCCTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCTG
    TTCTTATGGATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAAC
    TTCCTACCACTCACCCTAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCC
    CCTCAAACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTG
    GTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAG
    TGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCT
    CAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCC
    ACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCC
    TTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGA
    GCCTCATGATCTGCTGTCTGTAGTTCTGTGAGGTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCC
    TTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTA
    ACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGG
    AGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTC
    CAAGGAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAA
    AGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGT
    CACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAG
    TTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAAT
    ACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAG
    AAGCAGGGTTCTGGGTTTTGGAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTA
    CCTGCAGCTTTCTGGGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAG
    CTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGG
    GGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAA
    CATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGA
    ACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    54 opt_COX10*-ND1-3′UTR* ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACCATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCAT
    TCCTAATGCTTACCGAACGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGC
    CCCTACGGGCTACTACAACCCTTCGCTGACGCCATAAAACTCTTCACCAAAGAGCCCCTAAAACCCGC
    CACATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGAC
    CCCCCTCCCCATGCCCAACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTA
    GCCTAGCCGTTTACTCAATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCA
    CTGCGAGCAGTAGCCCAAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTA
    ATGAGTGGCTCCTTTAACCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATCA
    TGGCCCTTGGCCATGATGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGC
    CGAAGGGGAGTCCGAACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTC
    TTCATGGCCGAATACACAAACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAACA
    TATGACGCACTCTCCCCTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCTG
    TTCTTATGGATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAAC
    TTCCTACCACTCACCCTAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCC
    CCTCAAACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTG
    GTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAG
    TGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCT
    CAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCC
    ACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCC
    TTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGA
    GCCTCATGATCTGCTGTCTGTAGTTCTGTGAGGTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCC
    TTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTA
    ACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGG
    AGTCTCAAGCTGGACTGCCA
    55 opt_COX10*-opt_ND1-3′UTR ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACCATGGCCAACCTGCTGCTGCTGATCGTGCCCATCCTGATCGCCATGGCC
    TTCCTGATGCTGACCGAGCGCAAGATCCTGGGCTACATGCAGCTGCGCAAGGGCCCCAACGTGGTG
    GGCCCCTACGGCCTGCTGCAGCCCTTCGCCGACGCCATCAAGCTGTTCACCAAGGAGCCCCTGAAG
    CCCGCCACCAGCACCATCACCCTGTACATCACCGCCCCCACCCTGGCCCTGACCATCGCCCTGCTGC
    TGTGGACCCCCCTGCCCATGCCCAACCCCCTGGTGAACCTGAACCTGGGCCTGCTGTTCATCCTGGC
    CACCAGCAGCCTGGCCGTGTACAGCATCCTGTGGAGCGGCTGGGCCAGCAACAGCAACTACGCCCT
    GATCGGCGCCCTGCGCGCCGTGGCCCAGACCATCAGCTACGAGGTGACCCTGGCCATCATCCTGCT
    GAGCACCCTGCTGATGAGCGGCAGCTTCAACCTGAGCACCCTGATCACCACCCAGGAGCACCTGTGG
    CTGCTGCTGCCCAGCTGGCCCCTGGCCATGATGTGGTTCATCAGCACCCTGGCCGAGACCAACCGCA
    CCCCCTTCGACCTGGCCGAGGGCGAGAGCGAGCTGGTGAGCGGCTTCAACATCGAGTACGCCGCCG
    GCCCCTTCGCCCTGTTCTTCATGGCCGAGTACACCAACATCATCATGATGAACACCCTGACCACCACC
    ATCTTCCTGGGCACCACCTACGACGCCCTGAGCCCCGAGCTGTACACCACCTACTTCGTGACCAAGA
    CCCTGCTGCTGACCAGCCTGTTCCTGTGGATCCGCACCGCCTACCCCCGCTTCCGCTACGACCAGCT
    GATGCACCTGCTGTGGAAGAACTTCCTGCCCCTGACCCTGGCCCTGCTGATGTGGTACGTGAGCATG
    CCCATCACCATCAGCAGCATCCCCCCCCAGACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCT
    CCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGGTGGGTTTAGAACAA
    GATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAATATTACCCAAAATGC
    TCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGT
    CTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATAC
    ACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTG
    GCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCGTAGTTCTGTGAGCTCAGGTCCCTC
    AAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCA
    CACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTG
    GGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCA
    TTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACA
    CTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCC
    ACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTT
    TCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCA
    CATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCA
    CATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTG
    TGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGT
    CCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACT
    GGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAAT
    GTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGC
    TGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTAC
    AGCCTTCACATTTGTAGAAGCTTT
    56 opt_COX10*-opt_ND1-3′UTR* ATGGCCGCCAGCCCCCACACCCTGAGCAGCCGCCTGCTGACCGGCTGCGTGGGCGGCAGCGTGTG
    GTACCTGGAGCGCCGCACCATGGCCAACCTGCTGCTGCTGATCGTGCCCATCCTGATCGCCATGGCC
    TTCCTGATGCTGACCGAGCGCAAGATCCTGGGCTACATGCAGCTGCGCAAGGGCCCCAACGTGGTG
    GGCCCCTACGGCCTGCTGCAGCCCTTCGCCGACGCCATCAAGCTGTTCACCAAGGAGCCCCTGAAG
    CCCGCCACCAGCACCATCACCCTGTACATCACCGCCCCCACCCTGGCCCTGACCATCGCCCTGCTGC
    TGTGGACCCCCCTGCCCATGCCCAACCCCCTGGTGAACCTGAACCTGGGCCTGCTGTTCATCCTGGC
    CACCAGCAGCCTGGCCGTGTACAGCATCCTGTGGAGCGGCTGGGCCAGCAACAGCAACTACGCCCT
    GATCGGCGCCCTGCGCGCCGTGGCCCAGACCATCAGCTACGAGGTGACCCTGGCCATCATCCTGCT
    GAGCACCCTGCTGATGAGCGGCAGCTTCAACCTGAGCACCCTGATCACCACCCAGGAGCACCTGTGG
    CTGCTGCTGCCCAGCTGGCCCCTGGCCATGATGTGGTTCATCAGCACCCTGGCCGAGACCAACCGCA
    CCCCCTTCGACCTGGCCGAGGGCGAGAGCGAGCTGGTGAGCGGCTTCAACATCGAGTACGCCGCCG
    GCCCCTTCGCCCTGTTCTTCATGGCCGAGTACACCAACATCATCATGATGAACACCCTGACCACCACC
    ATCTTCCTGGGCACCACCTACGACGCCCTGAGCCCCGAGCTGTACACCACCTACTTCGTGACCAAGA
    CCCTGCTGCTGACCAGCCTGTTCCTGTGGATCCGCACCGCCTACCCCCGCTTCCGCTACGACCAGCT
    GATGCACCTGCTGTGGAAGAACTTCCTGCCCCTGACCCTGGCCCTGCTGATGTGGTACGTGAGCATG
    CCCATCACCATCAGCAGCATCCCCCCCCAGACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCT
    CCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAA
    GATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGC
    TCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGT
    CTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATAC
    ACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTG
    GCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTC
    AAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCA
    CACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTG
    GGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    57 COX8-ND4-3′UTR ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTGATGCTAAAACTAATCGTCCCAACAATTATGTTACTACCACTGACATGG
    CTTTCCAAAAAACACATGATTTGGATCAACACAACCACCCACAGCCTAATTATTAGCATCATCCCTCTA
    CTATTTTTTAACCAAATCAACAACAACCTATTTAGCTGTTCCCCAACCTTTTCCTCCGACCCCCTAACAA
    CCCCCCTCCTAATGCTAACTACCTGGCTCCTACCCCTCACAATCATGGCAAGCCAACGCCACTTATCC
    AGTGAACCACTATCACGAAAAAAACTCTACCTCTCTATGCTAATCTCCCTACAAATCTCCTTAATTATGA
    CATTCACAGCCACAGAACTAATCATGTTTTATATCTTCTTCGAAACCACACTTATCCCCACCTTGGCTAT
    CATCACCCGATGGGGCAACCAGCCAGAACGCCTGAACGCAGGCACATACTTCCTATTCTACACCCTA
    GTAGGCTCCCTTCCCCTACTCATCGCACTAATTTACACTCACAACACCCTAGGCTCACTAAACATTCTA
    CTACTCACTCTCACTGCCCAAGAACTATCAAACTCCTGGGCCAACAACTTAATGTGGCTAGCTTACACA
    ATGGCTTTTATGGTAAAGATGCCTCTTTACGGACTCCACTTATGGCTCCCTAAAGCCCATGTCGAAGCC
    CCCATCGCTGGGTCAATGGTACTTGCCGCAGTACTCTTAAAACTAGGCGGCTATGGTATGATGCGCCT
    CACACTCATTCTCAACCCCCTGACAAAACACATGGCCTACCCCTTCCTTGTACTATCCCTATGGGGCAT
    GATTATGACAAGCTCCATCTGCCTACGACAAACAGACCTAAAATCGCTCATTGCATACTCTTCAATCAG
    CCACATGGCCCTCGTAGTAACAGCCATTCTCATCCAAACCCCCTGGAGCTTCACCGGCGCAGTCATTC
    TCATGATCGCCCACGGGCTTACATCCTCATTACTATTCTGCCTAGCAAACTCAAACTACGAACGCACTC
    ACAGTCGCATCATGATCCTCTCTCAAGGACTTCAAACTCTACTCCCACTAATGGCTTTTTGGTGGCTTC
    TAGCAAGCCTCGCTAACCTCGCCTTACCCCCCACTATTAACCTACTGGGAGAACTCTCTGTGCTAGTA
    ACCACGTTCTCCTGGTCAAATATCACTCTCCTACTTACAGGACTCAACATGCTAGTCACAGCCCTATAC
    TCCCTCTACATGTTTACCACAACACAATGGGGCTCACTCACCCACCACATTAACAACATGAAACCCTCA
    TTCACACGAGAAAACACCCTCATGTTCATGCACCTATCCCCCATTCTCCTCCTATCCCTCAACCCCGAC
    ATCATTACCGGGTTTTCCTCTTAAGAGCACTGGGACGCCCACCGCCCCTTTcoCTCCGCTGCCAGGC
    GAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAA
    TTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAG
    AAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCT
    CTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCT
    TTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGC
    CAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGA
    GCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAAC
    CATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACAT
    GTTTGCCTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGC
    ATTTCAGAACTCCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCA
    GTTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTG
    TACCTCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTAT
    TGAGAAGGGAAGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCC
    TTGGGTGAAAAATACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGG
    CTTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTA
    CCAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCC
    AAAGGTCTTGAAGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGA
    TTCGATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGAT
    TGTAGGTAGATAACATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTA
    TTTACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATT
    TGTAGAAGCTTT
    58 COX8-ND4-3′UTR* ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTGATGCTAAAACTAATCGTCCCAACAATTATGTTACTACCACTGACATGG
    CTTTCCAAAAAACACATGATTTGGATCAACACAACCACCCACAGCCTAATTATTAGCATCATCCCTCTA
    CTATTTTTTAACCAAATCAACAACAACCTATTTAGCTGTTCCCCAACCTTTTCCTCCGACCCCCTAACAA
    CCCCCCTCCTAATGCTAACTACCTGGCTCCTACCCCTCACAATCATGGCAAGCCAACGCCACTTATCC
    AGTGAACCACTATCACGAAAAAAACTCTACCTCTCTATGCTAATCTCCCTACAAATCTCCTTAATTATGA
    CATTCACAGCCACAGAACTAATCATGTTTTATATCTTCTTCGAAACCACACTTATCCCCACCTTGGCTAT
    CATCACCCGATGGGGCAACCAGCCAGAACGCCTGAACGCAGGCACATACTTCCTATTCTACACCCTA
    GTAGGCTCCCTTCCCCTACTCATCGCACTAATTTACACTCACAACACCCTAGGCTCACTAAACATTCTA
    CTACTCACTCTCACTGCCCAAGAACTATCAAACTCCTGGGCCAACAACTTAATGTGGCTAGCTTACACA
    ATGGCTTTTATGGTAAAGATGCCTCTTTACGGACTCCACTTATGGCTCCCTAAAGCCCATGTCGAAGCC
    CCCATCGCTGGGTCAATGGTACTTGCCGCAGTACTCTTAAAACTAGGCGGCTATGGTATGATGCGCCT
    CACACTCATTCTCAACCCCCTGACAAAACACATGGCCTACCCCTTCCTTGTACTATCCCTATGGGGCAT
    GATTATGACAAGCTCCATCTGCCTACGACAAACAGACCTAAAATCGCTCATTGCATACTCTTCAATCAG
    CCACATGGCCCTCGTAGTAACAGCCATTCTCATCCAAACCCCCTGGAGCTTCACCGGCGCAGTCATTC
    TCATGATCGCCCACGGGCTTACATCCTCATTACTATTCTGCCTAGCAAACTCAAACTACGAACGCACTC
    ACAGTCGCATCATGATCCTCTCTCAAGGACTTCAAACTCTACTCCCACTAATGGCTTTTTGGTGGCTTC
    TAGCAAGCCTCGCTAACCTCGCCTTACCCCCCACTATTAACCTACTGGGAGAACTCTCTGTGCTAGTA
    ACCACGTTCTCCTGGTCAAATATCACTCTCCTACTTACAGGACTCAACATGCTAGTCACAGCCCTATAC
    TCCCTCTACATGTTTACCACAACACAATGGGGCTCACTCACCCACCACATTAACAACATGAAACCCTCA
    TTCACACGAGAAAACACCCTCATGTTCATGCACCTATCCCCCATTCTCCTCCTATCCCTCAACCCCGAC
    ATCATTACCGGGTTTTCCTCTTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGC
    GAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAA
    TTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAG
    AAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCT
    CTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCT
    TTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGC
    CAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGA
    GCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAAC
    CATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACAT
    GTTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    59 COX8-opt_ND4-3′UTR ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTGATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCTCTGACCTG
    GCTGAGCAAGAAACACATGATCTGGATCAACACCACCACGCACAGCCTGATCATCAGCATCATCCCTC
    TGCTGTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCTCTG
    ACAACACCTCTGCTGATGCTGACCACCTGGCTGCTGCCCCTCACAATCATGGCCTCTCAGAGACACCT
    GAGCAGCGAGCCCCTGAGCCGGAAGAAACTGTACCTGAGCATGCTGATCTCCCTGCAGATCTCTCTG
    ATCATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTTTTCGAGACAACGCTGATCCCCACA
    CTGGCCATCATCACCAGATGGGGCAACCAGCCTGAGAGACTGAACGCCGGCACCTACTTTCTGTTCT
    ACACCCTCGTGGGCAGCCTGCCACTGCTGATTGCCCTGATCTACACCCACAACACCCTGGGCTCCCT
    GAACATCCTGCTGCTGACACTGACAGCCCAAGAGCTGAGCAACAGCTGGGCCAACAATCTGATGTGG
    CTGGCCTACACAATGGCCTTCATGGTCAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCTAAAG
    CTCATGTGGAAGCCCCTATCGCCGGCTCTATGGTGCTGGCTGCAGTGCTGCTGAAACTCGGCGGCTA
    CGGCATGATGCGGCTGACCCTGATTCTGAATCCCCTGACCAAGCACATGGCCTATCCATTTCTGGTGC
    TGAGCCTGTGGGGCATGATTATGACCAGCAGCATCTGCCTGCGGCAGACCGATCTGAAGTCCCTGAT
    CGCCTACAGCTCCATCAGCCACATGGCCCTGGTGGTCACCGCCATCCTGATTCAGACCCCTTGGAGC
    TTTACAGGCGCCGTGATCCTGATGATTGCCCACGGCCTGACAAGCAGCCTGCTGTTTTGTCTGGCCAA
    CAGCAACTACGAGCGGACCCACAGCAGAATCATGATCCTGTCTCAGGGCCTGCAGACCCTCCTGCCT
    CTTATGGCTTTTTGGTGGCTGCTGGCCTCTCTGGCCAATCTGGCACTGCCTCCTACCATCAATCTGCT
    GGGCGAGCTGAGCGTGCTGGTCACCACATTCAGCTGGTCCAATATCACCCTGCTGCTCACCGGCCTG
    AACATGCTGGTTACAGCCCTGTACTCCCTGTACATGTTCACCACCACACAGTGGGGAAGCCTGACACA
    CCACATCAACAATATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCATCTGAGCCCCA
    TTCTGCTGCTGTCCCTGAATCCTGATATCATCACCGGCTTCTCCAGCTGAGAGCACTGGGACGCCCAC
    CGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCT
    GGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAAT
    ATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTT
    TCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATG
    GGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACAT
    GCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTG
    AGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTG
    GTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCT
    GTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTC
    CTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGCATCTTTATAGTTCAC
    GTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGG
    ATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTA
    CACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTGTGCTGGGCTAACC
    AGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGATATCTCCTGAATTC
    AGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTT
    ACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCC
    CACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTTTCGATTACTCAGT
    CTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACA
    GAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGCACTTATCTGA
    AATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAG
    CTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    60 COX8-opt_ND4-3′UTR* ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTGATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCTCTGACCTG
    GCTGAGCAAGAAACACATGATCTGGATCAACACCACCACGCACAGCCTGATCATCAGCATCATCCCTC
    TGCTGTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCTCTG
    ACAACACCTCTGCTGATGCTGACCACCTGGCTGCTGCCCCTCACAATCATGGCCTCTCAGAGACACCT
    GAGCAGCGAGCCCCTGAGCCGGAAGAAACTGTACCTGAGCATGCTGATCTCCCTGCAGATCTCTCTG
    ATCATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTTTTCGAGACAACGCTGATCCCCACA
    CTGGCCATCATCACCAGATGGGGCAACCAGCCTGAGAGACTGAACGCCGGCACCTACTTTCTGTTCT
    ACACCCTCGTGGGCAGCCTGCCACTGCTGATTGCCCTGATCTACACCCACAACACCCTGGGCTCCCT
    GAACATCCTGCTGCTGACACTGACAGCCCAAGAGCTGAGCAACAGCTGGGCCAACAATCTGATGTGG
    CTGGCCTACACAATGGCCTTCATGGTCAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCTAAAG
    CTCATGTGGAAGCCCCTATCGCCGGCTCTATGGTGCTGGCTGCAGTGCTGCTGAAACTCGGCGGCTA
    CGGCATGATGCGGCTGACCCTGATTCTGAATCCCCTGACCAAGCACATGGCCTATCCATTTCTGGTGC
    TGAGCCTGTGGGGCATGATTATGACCAGCAGCATCTGCCTGCGGCAGACCGATCTGAAGTCCCTGAT
    CGCCTACAGCTCCATCAGCCACATGGCCCTGGTGGTCACCGCCATCCTGATTCAGACCCCTTGGAGC
    TTTACAGGCGCCGTGATCCTGATGATTGCCCACGGCCTGACAAGCAGCCTGCTGTTTTGTCTGGCCAA
    CAGCAACTACGAGCGGACCCACAGCAGAATCATGATCCTGTCTCAGGGCCTGCAGACCCTCCTGCCT
    CTTATGGCTTTTTGGTGGCTGCTGGCCTCTCTGGCCAATCTGGCACTGCCTCCTACCATCAATCTGCT
    GGGCGAGCTGAGCGTGCTGGTCACCACATTCAGCTGGTCCAATATCACCCTGCTGCTCACCGGCCTG
    AACATGCTGGTTACAGCCCTGTACTCCCTGTACATGTTCACCACCACACAGTGGGGAAGCCTGACACA
    CCACATCAACAATATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCATCTGAGCCCCA
    TTCTGCTGCTGTCCCTGAATCCTGATATCATCACCGGCTTCTCCAGCTGAGAGCACTGGGACGCCCAC
    CGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCT
    GGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAAT
    ATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTT
    TCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATG
    GGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACAT
    GCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTG
    AGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTG
    GTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCT
    GTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    61 COX8-opt_ND4*-3′UTR ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTGATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCCCTGACCT
    GGCTGAGCAAGAAGCACATGATCTGGATCAACACCACCACCCACAGCCTGATCATCAGCATCATCCCC
    CTGCTGTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCCCT
    GACCACCCCCCTGCTGATGCTGACCACCTGGCTGCTGCCCCTGACCATCATGGCCAGCCAGCGCCAC
    CTGAGCAGCGAGCCCCTGAGCCGCAAGAAGCTGTACCTGAGCATGCTGATCAGCCTGCAGATCAGCC
    TGATCATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTCTTCGAGACCACCCTGATCCCC
    ACCCTGGCCATCATCACCCGCTGGGGCAACCAGCCCGAGCGCCTGAACGCCGGCACCTACTTCCTGT
    TCTACACCCTGGTGGGCAGCCTGCCCCTGCTGATCGCCCTGATCTACACCCACAACACCCTGGGCAG
    CCTGAACATCCTGCTGCTGACCCTGACCGCCCAGGAGCTGAGCAACAGCTGGGCCAACAACCTGATG
    TGGCTGGCCTACACCATGGCCTTCATGGTGAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCCA
    AGGCCCACGTGGAGGCCCCCATCGCCGGCAGCATGGTGCTGGCCGCCGTGCTGCTGAAGCTGGGC
    GGCTACGGCATGATGCGCCTGACCCTGATCCTGAACCCCCTGACCAAGCACATGGCCTACCCCTTCC
    TGGTGCTGAGCCTGTGGGGCATGATCATGACCAGCAGCATCTGCCTGCGCCAGACCGACCTGAAGAG
    CCTGATCGCCTACAGCAGCATCAGCCACATGGCCCTGGTGGTGACCGCCATCCTGATCCAGACCCCC
    TGGAGCTTCACCGGCGCCGTGATCCTGATGATCGCCCACGGCCTGACCAGCAGCCTGCTGTTCTGCC
    TGGCCAACAGCAACTACGAGCGCACCCACAGCCGCATCATGATCCTGAGCCAGGGCCTGCAGACCCT
    GCTGCCCCTGATGGCCTTCTGGTGGCTGCTGGCCAGCCTGGCCAACCTGGCCCTGCCCCCCACCAT
    CAACCTGCTGGGCGAGCTGAGCGTGCTGGTGACCACCTTCAGCTGGAGCAACATCACCCTGCTGCTG
    ACCGGCCTGAACATGCTGGTGACCGCCCTGTACAGCCTGTACATGTTCACCACCACCCAGTGGGGCA
    GCCTGACCCACCACATCAACAACATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCAC
    CTGAGCCCCATCCTGGTGCTGAGCCTGAACCCCGACATCATCACCGGCTTCAGCAGCTAAGAGCACT
    GGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGA
    AGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTT
    TTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAA
    AAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCCTATTCTGTTTCT
    TCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACG
    CACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTC
    TGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGG
    CCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAG
    GACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTG
    CCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGCAT
    CTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTT
    AATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCC
    ACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTGTG
    CTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGATA
    TCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGA
    ATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTC
    CTTTGTGCTCCCACGGGTCTACAGAGTCCCAlCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTTTC
    GATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACA
    ACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGC
    ACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTC
    TGGAAAAAGCTTCTACAACTTGTTACAGCCTGTCACATTTTAGAAGCTTT
    62 COX8-opt_ND4*-3′UTR* ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTGATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCCCTGACCT
    GGCTGAGCAAGAAGCACATGATCTGGATCAACACCACCACCCACAGCCTGATCATCAGCATCATCCCC
    CTGCTGTTCTTCAACCAGATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCCCT
    GACCACCCCCCTGCTGATGCTGACCACCTGGCTGCTGCCCCTGACCATCATGGCCAGCCAGCGCCAC
    CTGAGCAGCGAGCCCCTGAGCCGCAAGAAGCTGTACCTGAGCATGCTGATCAGCCTGCAGATCAGCC
    TGATCATGACCTTCACCGCCACCGAGCTGATCATGTTCTACATCTTCTTCGAGACCACCCTGATCCCC
    ACCCTGGCCATCATCACCCGCTGGGGCAACCAGCCCGAGCGCCTGAACGCCGGCACCTACTTCCTGT
    TCTACACCCTGGTGGGCAGCCTGCCCCTGCTGATCGCCCTGATCTACACCCACAACACCCTGGGCAG
    CCTGAACATCCTGCTGCTGACCCTGACCGCCCAGGAGCTGAGCAACAGCTGGGCCAACAACCTGATG
    TGGCTGGCCTACACCATGGCCTTCATGGTGAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCCA
    AGGCCCACGTGGAGGCCCCCATCGCCGGCAGCATGGTGCTGGCCGCCGTGCTGCTGAAGCTGGGC
    GGCTACGGCATGATGCGCCTGACCCTGATCCTGAACCCCCTGACCAAGCACATGGCCTACCCCTTCC
    TGGTGCTGAGCCTGTGGGGCATGATCATGACCAGCAGCATCTGCCTGCGCCAGACCGACCTGAAGAG
    CCTGATCGCCTACAGCAGCATCAGCCACATGGCCCTGGTGGTGACCGCCATCCTGATCCAGACCCCC
    TGGAGCTTCACCGGCGCCGTGATCCTGATGATCGCCCACGGCCTGACCAGCAGCCTGCTGTTCTGCC
    TGGCCAACAGCAACTACGAGCGCACCCACAGCCGCATCATGATCCTGAGCCAGGGCCTGCAGACCCT
    GCTGCCCCTGATGGCCTTCTGGTGGCTGCTGGCCAGCCTGGCCAACCTGGCCCTGCCCCCCACCAT
    CAACCTGCTGGGCGAGCTGAGCGTGCTGGTGACCACCTTCAGGTGGAGCAACATCACCCTGCTGCTG
    ACCGGCCTGAACATGCTGGTGACCGCCCTGTACAGCCTGTACATGTTCACCACCACCCAGTGGGGCA
    GCCTGACCCACCACATCAACAACATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCAC
    CTGAGCCCCATCCTGCTGCTGAGCCTGAACCCCGACATCATCACCGGCTTCAGCAGCTAAGAGCACT
    GGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGA
    AGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTT
    TTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAA
    AAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCT
    TCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACG
    CACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTC
    TGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGG
    CCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAG
    GACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTG
    CCA
    63 COX8-ND6-3′UTR ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTGATGATGTATGCTTTGTTTCTGTTGAGTGTGGGTTTAGTAATGGGGTTT
    GTGGGGTTTTCTTCTAAGCCTTCTCCTATTTATGGGGGTTTAGTATTGATTGTTAGCGGTGTGGTCGGG
    TGTGTTATTATTCTGAATTTTGGGGGAGGTTATATGGGTTTAATGGTTTTTTTAATTTATTTAGGGGGAA
    TGATGGTTGTCTTTGGATATACTACAGCGATGGCTATTGAGGAGTATCCTGAGGCATGGGGGTCAGGG
    GTTGAGGTCTTGGTGAGTGTTTTAGTGGGGTTAGCGATGGAGGTAGGATTGGTGCTGTGGGTGAAAG
    AGTATGATGGGGTGGTGGTTGTGGTAAACTTTAATAGTGTAGGAAGCTGGATGATTTATGAAGGAGAG
    GGGTCAGGGTTGATTCGGGAGGATCCTATTGGTGCGGGGGCTTTGTATGATTATGGGCGTTGGTTAG
    TAGTAGTTACTGGTTGGACATTGTTTGTTGGTGTATATATTGTAATTGAGATTGCTCGGGGGAATTAGG
    AGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAAC
    ACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACA
    GTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATA
    CAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTG
    TTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACC
    ACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTG
    CTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCC
    AGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAG
    CTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGG
    ACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAG
    GCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGG
    ACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGT
    CGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGG
    GTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCC
    TGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCT
    GGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTT
    TAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATG
    TTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTT
    AAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACT
    GTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGG
    AATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    64 COX8-ND6-3′UTR* ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTGATGATGTATGCTTTGTTTCTGTTGAGTGTGGGTTTAGTAATGGGGTTT
    GTGGGGTTTTCTTCTAAGCCTTCTCCTATTTATGGGGGTTTAGTATTGATTGTTAGCGGTGTGGTCGGG
    TGTGTTATTATTCTGAATTTTGGGGGAGGTTATATGGGTTTAATGGTTTTTTTAATTTATTTAGGGGGAA
    TGATGGTTGTCTTTGGATATACTACAGCGATGGCTATTGAGGAGTATCCTGAGGCATGGGGGTCAGGG
    GTTGAGGTCTTGGTGAGTGTTTTAGTGGGGTTAGCGATGGAGGTAGGATTGGTGCTGTGGGTGAAAG
    AGTATGATGGGGTGGTGGTTGTGGTAAACTTTAATAGTGTAGGAAGCTGGATGATTTATGAAGGAGAG
    GGGTCAGGGTTGATTCGGGAGGATCCTATTGGTGCGGGGGCTTTGTATGATTATGGGCGTTGGTTAG
    TAGTAGTTACTGGTTGGACATTGTTTGTTGGTGTATATATTGTAATTGAGATTGCTCGGGGGAATTAGG
    AGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAAC
    ACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACA
    GTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATA
    CAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTG
    TTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACC
    ACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTG
    CTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCC
    AGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAG
    CTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGG
    ACTGCCA
    65 COX8-opt_ND6-3′UTR ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTGATGATGTACGCCCTGTTCCTGCTGAGCGTGGGCCTGGTGATGGGCT
    TCGTGGGCTTCAGCAGCAAGCCCAGCCCCATCTACGGCGGCCTGGTGCTGATCGTGAGCGGCGTGG
    TGGGCTGCGTGATCATCCTGAACTTCGGCGGCGGCTACATGGGCCTGATGGTGTTCCTGATCTACCT
    GGGCGGCATGATGGTGGTGTTCGGCTACACCACCGCCATGGCCATCGAGGAGTACCCCGAGGCCTG
    GGGCAGCGGCGTGGAGGTGCTGGTGAGCGTGCTGGTGGGCCTGGCCATGGAGGTGGGCCTGGTGC
    TGTGGGTGAAGGAGTACGACGGCGTGGTGGTGGTGGTGAACTTCAACAGCGTGGGCAGCTGGATGA
    TCTACGAGGGCGAGGGCAGCGGCCTGATCCGCGAGGACCCCATCGGCGCCGGCGCCCTGTACGAC
    FACGGCCGCTGGCTGGTGGTGGTGACCGGCTGGACCCTGTTCGTGGGCGTGTACATCGTGATCGAG
    ATCGCCCGCGGCAACTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCA
    TGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGG
    TGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGC
    ATCAGCTCAGTCAGTGAATACAAAAAAGGAATTTATTTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTC
    CAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGT
    TCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAA
    AGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACC
    CCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGT
    CCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGC
    CTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCA
    GAACTCCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCT
    TCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTC
    TGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAA
    GGGAAGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGT
    GAAAAATACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAA
    GAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAAT
    ACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGT
    CTTGAAGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGAT
    TGGTCGGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAG
    GTAGATAACATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTAC
    TGTGGAGAACATTGCATAGGAATGTCTGGAAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAG
    AAGCTTT
    66 COX8-opt_ND6-3′UTR ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTGATGATGTACGCCCTGTTCCTGCTGAGCGTGGGCCTGGTGATGGGCT
    TCGTGGGCTTCAGCAGCAAGCCCAGCCCCATCTACGGCGGCCTGGTGCTGATCGTGAGCGGCGTGG
    TGGGCTGCGTGATCATCCTGAACTTCGGCGGCGGCTACATGGGCCTGATGGTGTTCCTGATCTACCT
    GGGCGGCATGATGGTGGTGTTCGGCTACACCACCGCCATGGCCATCGAGGAGTACCCCGAGGCCTG
    GGGCAGCGGCGTGGAGGTGCTGGTGAGCGTGCTGGTGGGCCTGGCCATGGAGGTGGGCCTGGTGC
    TGTGGGTGAAGGAGTACGACGGCGTGGTGGTGGTGGTGAACTTCAACAGCGTGGGCAGCTGGATGA
    TCTACGAGGGCGAGGGCAGCGGCCTGATCCGCGAGGACCCCATCGGCGCCGGCGCCCTGTACGAC
    TACGGCCGCTGGCTGGTGGTGGTGACCGGCTGGACCCTGTTCGTGGGCGTGTACATCGTGATCGAG
    ATCGCCCGCGGCAACTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCA
    TGTTGTGGTAATTCTGGAACACAAGATGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGG
    TGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGC
    ATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTC
    CAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGT
    TCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAA
    AGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACC
    CCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGT
    CCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGC
    CTTGGGAGTCTCAAGCTGGACTGCCA
    67 COX8-ND1-3′UTR ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTGATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGC
    ATTCCTAATGCTTACCGAACGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGG
    CCCCTACGGGCTACTACAACCCTTCGCTGACGCCATAAAACTCTTCACCAAAGAGCCCCTAAAACCCG
    CCACATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGA
    CCCCCCTCCCCATGCCCAACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCT
    AGCCTAGCCGTTTACTCAATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGC
    ACTGCGAGCAGTAGCCCAAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACT
    AATGAGTGGCTCCTTTAACCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATC
    ATGGCCCTTGGCCATGATGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTG
    CCGAAGGGGAGTCCGAACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATT
    CTTCATGGCCGAATACACAAACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAAC
    ATATGACGCACTCTCCCCTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCT
    GTTCTTATGGATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAA
    ACTTCCTACCACTCACCCTAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTC
    CCCCTCAAACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTG
    TGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTC
    AGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAG
    CTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACC
    CCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCAT
    CCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGT
    GAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTT
    CCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTC
    TAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGG
    GAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACT
    CCAAGGAGTCACAGGCATCTTTATAGTTCACGTCATATAGACACTGTTGGAAGCAGTTCCTTCTAA
    AAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGA
    GTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGA
    AGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAA
    AATACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGC
    CAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGG
    TTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGA
    AGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTC
    GGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGAT
    AACATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGA
    GAACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTT
    T
    68 COX8-ND1-3′UTR* ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTGATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAAATCGCAATGGC
    ATTCCTAATGCTTACCGAACGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGG
    CCCCTACGGGCTACTACAACCCTTCGCTGACGCCATAAAACTCTTCACCAAAGAGCCCCTAAAACCCG
    CCACATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGA
    CCCCCCTCCCCATGCCCAACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCT
    AGCCTAGCCGTTTACTCAATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGC
    ACTGCGAGCAGTAGCCCAAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACT
    AATGAGTGGCTCCTTTAACCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATC
    ATGGCCCTTGGCCATGATGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTG
    CCGAAGGGGAGTCCGAACTAGTCTCAGGCTTCAAACATCGAATACGCCGCAGGCCCCTTCGCCCTATT
    CTTCATGGCCGAATACACAAACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAAC
    ATATGACGCACTCTCCCCTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCT
    GTTCTTATGGATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAA
    ACTTCCTACCACTCACCCTAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTC
    CCCCTCAAACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTG
    TGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTC
    AGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAG
    CTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACC
    CCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCAT
    CCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGT
    GAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTT
    CCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTC
    TAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGG
    GAGTCTCAAGCTGGACTGCCA
    69 COX8-opt_ND1-3′UTR ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTGATGGCCAACCTGCTGCTGCTGATCGTGCCCATCCTGATCGCCATGG
    CCTTCCTGATGCTGACCGAGCGCAAGATCCTGGGCTACATGCAGCTGCGCAAGGGCCCCAACGTGGT
    GGGCCCCTACGGCCTGCTGCAGCCCTTCGCCGACGCCATCAAGCTGTTCACCAAGGAGCCCCTGAA
    GCCCGCCACCAGCACCATCACCCTGTACATCACCGCCCCCACCCTGGCCCTGACCATCGCCCTGCTG
    CTGTGGACCCCCCTGCCCATGCCCAACCCCCTGGTGAACCTGAACCTGGGCCTGCTGTTCATCCTGG
    CCACCAGCAGCCTGGCCGTGTACAGCATCCTGTGGAGCGGCTGGGCCAGCAACAGCAACTACGCCC
    TGATCGGCGCCCTGCGCGCCGTGGCCCAGACCATCAGCTACGAGGTGACCCTGGCCATCATCCTGC
    TGAGCACCCTGCTGATGAGCGGCAGCTTCAACCTGAGCACCCTGATCACCACCCAGGAGCACCTGTG
    GCTGCTGCTGCCCAGCTGGCCCCTGGCCATGATGTGGTTCATCAGCACCCTGGCCGAGACCAACCG
    CACCCCCTTCGACCTGGCCGAGGGCGAGAGCGAGCTGGTGAGCGGCTTCAACATCGAGTACGCCGC
    CGGCCCCTTCGCCCTGTTCTTCATGGCCGAGTACACCAACATCATCATGATGAACACCCTGACCACCA
    CCATCTTCCTGGGCACCACCTACGACGCCCTGAGCCCCGAGCTGTACACCACCTACTTCGTGACCAA
    GACCCTGCTGCTGACCAGCCTGTTCCTGTGGATCCGCACCGCCTACCCCCGCTTCCGCTACGACCAG
    CTGATGCACCTGCTGTGGAAGAACTTCCTGCCCCTGACCCTGGCCCTGCTGATGTGGTACGTGAGCA
    TGCCCATCACCATCAGCAGCATCCCCCCCCAGACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCC
    CTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAAC
    AAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAAT
    GCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGG
    GTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACAT
    ACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAG
    TGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCC
    TCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCC
    CACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGAC
    TGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCC
    CATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGA
    CACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCC
    CCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTT
    TTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCT
    CACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTC
    CACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGG
    TGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGA
    GTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTA
    CTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAA
    ATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTG
    GCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAAACTTGTT
    ACAGCCTTCACATTTGTAGAAGCTTT
    70 COX8-opt_ND1-3′UTR* ATGTCCGTCCTGACGCGCCTGCTGCTGCGGGGCTTGACACGGCTCGGCTCGGCGGCTCCAGTGCGG
    CGCGCCAGAATCCATTCGTTGATGGCCAACCTGCTGCTGCTGATCGTGCCCATCCTGATCGCCATGG
    CCTTCCTGATGCTGACCGAGCGCAAGATCCTGGGCTACATGCAGCTGCGCAAGGGCCCCAACGTGGT
    GGGCCCCTACGGCCTGCTGCAGCCCTTCGCCGACGCCATCAAGCTGTTCACCAAGGAGCCCCTGAA
    GCCCGCCACCAGCACCATCACCCTGTACATCACCGCCCCCACCCTGGCCCTGACCATCGCCCTGCTG
    CTGTGGACCCCCCTGCCCATGCCCAACCCCCTGGTGAACCTGAACCTGGGCCTGCTGTTCATCCTGG
    CCACCAGCAGCCTGGCCGTGTACAGCATCCTGTGGAGCGGCTGGGCCAGCAACAGCAACTACGCCC
    TGATCGGCGCCCTGCGCGCCGTGGCCCAGACCATCAGCTACGAGGTGACCCTGGCCATCATCCTGC
    TGAGCACCCTGCTGATGAGCGGCAGCTTCAACCTGAGCACCCTGATCACCACCCAGGAGCACCTGTG
    GCTGCTGCTGCCCAGCTGGCCCCTGGCCATGATGTGGTTCATCAGCACCCTGGCCGAGACCAACCG
    CACCCCCTTCGACCTGGCCGAGGGCGAGAGCGAGCTGGTGAGCGGCTTCAACATCGAGTACGCCGC
    CGGCCCCTTCGCCCTGTTCTTCATGGCCGAGTACACCAACATCATCATGATGAACACCCTGACCACCA
    CCATCTTCCTGGGCACCACCTACGACGCCCTGAGCCCCGAGCTGTACACCACCTACTTCGTGACCAA
    GACCCTGCTGCTGACCAGCCTGTTCCTGTGGATCCGCACCGCCTACCCCCGCTTCCGCTACGACCAG
    CTGATGCACCTGCTGTGGAAGAACTTCCTGCCCCTGACCCTGGCCCTGCTGATGTGGTACGTGAGCA
    TGCCCATCACCATCAGCAGCATCCCCCCCCAGACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCC
    CTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAAC
    AAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAAT
    GCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGAATTATTTTTCCCTTTGAGG
    GTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACAT
    ACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAG
    TGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCC
    TCAAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCC
    CACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGAC
    TGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    71 CPA1-ND4-3′UTR GTGCTGCCCGCCTAGAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTGATGCTAAAACTAATCGTCCCAACAATTATGTTACTACCACTGACATGGCTTTCCAAAAAACACATG
    ATTTGGATCAACACAACCACCCACAGCCTAATTATTAGCATCATCCCTCTACTATTTTTTAACCAAATCA
    ACAACAACCTATTTAGCTGTTCCCCAACCTTTTCCTCCGACCCCCTAACAACCCCCCTCCTAATGCTAA
    CTACCTGGCTCCTACCCCTCACAATCATGGCAAGCCAACGCCACTTATCCAGTGAACCACTATCACGA
    AAAAAACTCTACCTCTCTATGCTAATCTCCCTACAAATCTCCTTAATTATGACATTCACAGCCACAGAAC
    TAATCATGTTTTATATCTTCTTCGAAACCACACTTATCCCCACCTTGGCTATCATCACCCGATGGGGCA
    ACCAGCCAGAACGCCTGAACGCAGGCACATACTTCCTATTCTACACCCTAGTAGGCTCCCTTCCCCTA
    CTCATCGCACTAATTTACACTCACAACACCCTAGGCTCACTAAACATTCTACTACTCACTCTCACTGCC
    CAAGAACTATCAAACTCCTGGGCCAACAACTTAATGTGGCTAGCTTACACAATGGCTTTTATGGTAAAG
    ATGCCTCTTTACGGACTCCACTTATGGCTCCCTAAAGCCCATGTCGAAGCCCCCATCGCTGGGTCAAT
    GGTACTTGCCGCAGTACTCTTAAAACTAGGCGGCTATGGTATGATGCGCCTCACACTCATTCTCAACC
    CCCTGACAAAAACACATGGCCTACCCCTTCCTTGTACTATCCCTATGGGGCATGATTATGACAAGGTCC
    ATCTGCCTACGACAAACAGACCTAAAATCGCTCATTGCATACTCTTCAATCAGCCACATGGCCCTCGTA
    GTAACAGCCATTCTCATCCAAACCCCCTGGAGCTTCACCGGCGCAGTCATTCTCATGATCGCCCACGG
    GCTTACATCCTCATTACTATTCTGCCTAGCAAACTCAAACTACGAACGCACTCACAGTCGCATCATGAT
    CCTCTCTCAAGGACTTCAAACTCTACTCCCACTAATGGCTTTTTGGTGGCTTCTAGCAAGCCTCGCTAA
    CCTCGCCTTACCCCCCACTATTAACCTACTGGGAGAACTCTCTGTGCTAGTAACCACGTTCTCCTGGT
    CAAATATCACTCTCCTACTTACAGGACTCAACATGCTAGTCACAGCCCTATACTCCCTCTACATGTTTA
    CCACAACACAATGGGGCTCACTCACCCACCACATTAACAACATGAAACCCTCATTCACACGAGAAAAC
    ACCCTCATGTTCATGCACCTATCCCCCATTCTCCTCCTATCCCTCAACCCCGACATCATTACCGGGTTT
    TCCTCTTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTA
    ATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGA
    TCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAG
    TCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACC
    CTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTAC
    CACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAAGTGTGAGCCT
    CATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGT
    GACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAA
    TACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTC
    TCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAG
    GAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGG
    TAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACT
    ACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAG
    GAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACAT
    GTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTTTAAGCCAGAAGC
    AGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTG
    CAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTG
    ACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTA
    GGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATC
    CAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACAT
    TGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    72 CPA1-ND4-3′UTR-2* GTGCTGCCCGCCTAGAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTGATGCTAAAACTAATCGTCCCAACAATTATGTTACTACCACTGACATGGCTTTCCAAAAAACACATG
    ATTTGGATCAACACAACCACCCACAGCCTAATTATTAGCATCATCCCTCTACTATTTTTTAACCAAATCA
    ACAACAACCTATTTAGCTGTTCCCCAACCTTTTCCTCCGACCCCCTAACAACCCCCCTCCTAATGCTAA
    CTACCTGGCTCCTACCCCTCACAATCATGGCAAGCCAACGCCACTTATCCAGTGAACCACTATCACGA
    AAAAAACTCTACCTCTCTATGCTAATCTCCCTACAAATCTCCTTAATTATGACATTCACAGCCACAGAAC
    TAATCATGTTTTATATCTTCTTCGAAACCACACTTATCCCCACCTTGGCTATCATCACCCGATGGGGCA
    ACCAGCCAGAACGCCTGAACGCAGGCACATACTTCCTATTCTACACCCTAGTAGGCTCCCTTCCCCTA
    CTCATCGCACTAATTTACACTCACAACACCCTAGGCTCACTAAACATTCTACTACTCACTCTCACTGCC
    CAAGAACTATCAAACTCCTGGGCCAACAACTTAATGTGGCTAGCTTACACAATGGCTTTTATGGTAAAG
    ATGCCTCTTTACGGACTCCACTTATGGCTCCCTAAAGCCCATGTCGAAGCCCCCATCGCTGGGTCAAT
    GGTACTTGCCGCAGTACTCTTAAAACTAGGCGGCTATGGTATGATGCGCCTCACACTCATTCTCAACC
    CCCTGACAAAACACATGGCCTACCCCTTCCTTGTACTATCCCTATGGGGCATGATTATGACAAGCTCC
    ATCTGCCTACGACAAACAGACCTAAAATCGCTCATTGCATACTCTTCAATCAGCCACATGGCCCTCGTA
    GTAACAGCCATTCTCATCCAAACCCCCTGGAGCTTCACCGGCGCAGTCATTCTCATGATCGCCCACGG
    GCTTACATCCTCATTACTATTCTGCCTAGCAAACTCAAACTACGAACGCACTCACAGTCGCATCATGAT
    CCTCTCTCAAGGACTTCAAACTCTACTCCCACTAATGGCTTTTTGGTGGCTTCTAGCAAGCCTCGCTAA
    CCTCGCCTTACCCCCCACTATTAACCTACTGGGAGAACTCTCTGTGCTAGTAACCACGTTCTCCTGGT
    CAAATATCACTCTCCTACTTACAGGACTCAACATGCTAGTCACAGCCCTATACTCCCTCTACATGTTTA
    CCACAACACAATGGGGCTCACTCACCCACCACATTAACAACATGAAACCCTCATTCACACGAGAAAAC
    ACCCTCATGTTCATGCACCTATCCCCCATTCTCCTCCTATCCCTCAACCCCGACATCATTACCGGGTTT
    TCCTCTTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTA
    ATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGA
    TCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAG
    TCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACC
    CTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTAC
    CACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCT
    CATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGT
    GACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAA
    TACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTC
    TCAAGCTGGACTGCCA
    73 OPA1-opt_ND4-3′UTR GTGCTGCCCGCCTAGAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTGATGCTGAAAGCTGATCGTGCCCACCATCATGCTGCTGCCTCTGACCTGGCTGAGCAAGAAACACAT
    GATCTGGATCAACACCACCACGCACAGCCTGATCATCAGCATCATCCCTCTGCTGTTCTTCAACCAGA
    TCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCTCTGACAACACCTCTGCTGATG
    CTGACCACCTGGCTGCTGCCCCTCACAATCATGGCCTCTCAGAGACACCTGAGCAGCGAGCCCCTGA
    GCCGGAAAGAAACTGTACCTGAGCATGCTGATCTCCCTGCAGATCTCTCTGATCATGACCTTCACCGCC
    ACCGAGCTGATCATGTTCTACATCTTTTTCGAGACAACGCTGATCCCCACACTGGCCATCATCACCAG
    ATGGGGCAACCAGCCTGAGAGACTGAACGCCGGCACCTACTTTCTGTTCTACACCCTCGTGGGCAGC
    CTGCCACTGCTGATTGCCCTGATCTACACCCACAACACCCTGGGCTCCCTGAACATCCTGCTGCTGAC
    ACTGACAGCCCAAGAGCTGAGCAACAGCTGGGCCAACAATCTGATGTGGCTGGCCTACACAATGGCC
    TTCATGGTCAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCTAAAGCTCATGTGGAAGCCCCTAT
    CGCCGGCTCTATGGTGCTGGCTGCAGTGCTGCTGAAACTCGGCGGCTACGGCATGATGCGGCTGAC
    CCTGATTCTGAATCCCCTGACCAAGCACATGGCCTATCCATTTCTGGTGCTGAGCCTGTGGGGCATGA
    TTATGACCAGCAGCATCTGCCTGCGGCAGACCGATCTGAAGTCCCTGATCGCCTACAGCTCCATCAG
    CCACATGGCCCTGGTGGTCACCGCCATCCTGATTCAGACCCCTTGGAGCTTTACAGGCGCCGTGATC
    CTGATGATTGCCCACGGCCTGACAAGCAGCCTGCTGTTTTGTCTGGCCAACAGCAACTACGAGCGGA
    CCCACAGCAGAATCATGATCCTGTCTCAGGGCCTGCAGACCCTCCTGCCTCTTATGGCTTTTTGGTGG
    CTGCTGGCCTCTCTGGCCAATCTGGCACTGCCTCCTACCATCAATCTGCTGGGCGAGCTGAGCGTGC
    TGGTCACCACATTCAGGTGGTCCAATATCACCCTGCTGCTCACCGGCCTGAACATGCTGGTTACAGCC
    CTGTACTCCCTGTACATGTTCACCACCACACAGTGGGGAAGCCTGACACACCACATCAACAATATGAA
    GCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCATCTGAGCCCCATTCTGCTGCTGTCCCTGA
    ATCCTGATATCATCACCGGCTTCTCCAGCTGAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGC
    TGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATT
    ATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATTGCTCC
    CCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTT
    TATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACA
    GCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCA
    CTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAA
    GCCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACA
    CATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGG
    GATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATT
    GCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACT
    GTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCAC
    CCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTC
    AAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACA
    TTCCTGTCCCTTGGGTGTAAAAATACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACA
    TGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTG
    GTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTC
    CCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTG
    GTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGT
    CTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTG
    CCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAG
    CCTTCACATTTGTAGAAGCTTT
    74 OPA1-opt_ND4-3′UTFR* GTGCTGCCCGCCTAGAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTGATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCTCTGACCTGGCTGAGCAAGAAACACAT
    GATCTGGATCAACACCACCACGCACAGCCTGATCATCAGCATCATCCCTCTGCTGTTCTTCAACCAGA
    TCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCTCTGACAACACCTCTGCTGATG
    CTGACCACCTGGCTGCTGCCCCTCACAATCATGGCCTCTCAGAGACACCTGAGCAGCGAGCCCCTGA
    SCCGGAGAAACTSTACCTGAGCATSCTSATCTCCCTGCAGATCTCTCTGATCATSACCTTCACCCCC
    ACCGAGCTGATCATGTTCTACATCTTTTTCGAGACAACGCTGATCCCCACACTGGCCATCATCACCAG
    ATGGGGCAACCAGCCTGAGAGACTGAACGCCGGCACCTACTTTCTGTTCTACACCCTCGTGGGCAGC
    CTGCCACTGCTGATTGCCCTGATCTACACCCACAACACCCTGGGCTCCCTGAACATCCTGCTGCTGAC
    ACTGACAGCCCAAGAGCTGAGCAACAGCTGGGCCAACAATCTGATGTGGCTGGCCTACACAATGGCC
    TTCATGGTCAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCTAAAGCTCATGTGGAAGCCCCTAT
    CGCCGGCTCTATGGTGCTGGCTGCAGTGCTGCTGAAACTCGGCGGCTACGGCATGATGCGGCTGAC
    CCTGATTCTGAATCCCCTGACCAAGCACATGGCCTATCCATTTCTGGTGCTGAGCCTGTGGGGCATGA
    TTATGACCAGCAGCATCTGCCTGCGGCAGACCGATCTGAAGTCCCTGATCGCCTACAGCTCCATCAG
    CCACATGGCCCTGGTGGTCACCGCCATCCTGATTCAGACCCCTTGGAGCTTTACAGGCGCCGTGATC
    CTGATGATTGCCCACGGCCTGACAAGCAGCCTGCTGTTTTGTCTGGCCAACAGCAACTACGAGCGGA
    CCCACAGCAGAATCATGATCCTGTCTCAGGGCCTGCAGACCCTCCTGCCTCTTATGGCTTTTTGGTGG
    CTGCTGGCCTCTCTGGCCAATCTGGCACTGCCTCCTACCATCAATCTGCTGGGCGAGCTGAGCGTGC
    TGGTCACCACATTCAGCTGGTCCAATATCACCCTGCTGCTCACCGGCCTGAACATGCTGGTTACAGCC
    CTGTACTCCCTGTACATGTTCACCACCACACAGTGGGGAAGCCTGACACACCACATCAACAATATGAA
    GCCCAGCTTCACCCGCGAGAAACACCCTGATGTTCATGCATCTGAGCCCCATTCTGCTGCTGTCCCTGA
    ATCCTGATATCATCACCGGCTTCTCCAGCTGAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGC
    TGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATT
    ATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCC
    TGCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTT
    TATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTTCTTCCTCCTCACATGGGGGTACACATACACA
    GCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCA
    CTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAA
    GGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACA
    CATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGG
    GATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    75 OPA1-opt_ND4*-3′UTR GTGCTGCCCGCCTAGAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTGATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCCCTGACCTGGCTGAGCAAGAAGCACA
    TGATCTGGATCAACACCACCACCCACAGCCTGATCATCAGCATCATCCCCCTGCTGTTCTTCAACCAG
    ATCAACAACAACCTGTTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCCCTGACCACCCCCCTGCTGA
    TGCTGACCACCTGGCTGCTGCCCCTGACCATCATGGCCAGCCAGCGCCACCTGAGCAGCGAGCCCC
    TGAGCCGCAAGAAGCTGTACCTGAGCATGCTGATCAGCCTGCAGATCAGCCTGATCATGACCTTCACC
    GCCACCGAGCTGATCATGTTCTACATCTTCTTCGAGACCACCCTGATCCCCACCCTGGCCATCATCAC
    CCGCTGGGGCAACCAGCCCGAGCGCCTGAACGCCGGCACCTACTTCCTGTTCTACACCCTGGTGGG
    CAGCCTGCCCCTGCTGATCGCCCTGATCTACACCCACAACACCCTGGGCAGCCTGAACATCCTGCTG
    CTGACCCTGACCGCCCAGGAGCTGAGCAACAGCTGGGCCAACAACCTGATGTGGCTGGCCTACACCA
    TGGCCTTCATGGTGAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCCAAGGCCCACGTGGAGG
    CCCCCATCGCCGGCAGCATGGTGCTGGCCGCCGTGCTGCTGAAGCTGGGCGGCTACGGCATGATGC
    GCCTGACCCTGATCCTGAACCCCCTGACCAAGCACATGGCCTACCCCTTCCTGGTGCTGAGCCTGTG
    GGGCATGATCATGACCAGCAGCATCTGCCTGCGCCAGACCGACCTGAAGAGCCTGATCGCCTACAGC
    AGCATCAGCCACATGGCCCTGGTGGTGACCGCCATCCTGATCCAGACCCCCTGGAGCTTCACCGGCG
    CCGTGATCCTGATGATCGCCCACGGCCTGACCAGCAGCCTGCTGTTCTGCCTGGCCAACAGCAACTA
    CGAGCGCACCCACAGCCGCATCATGATCCTGAGCCAGGGCCTGCAGACCCTGCTGCCCCTGATGGC
    CTTCTGGTGGCTGCTGGCCAGCCTGGCCAACCTGGCCCTGCCCCCCACCATCAACCTGCTGGGCGA
    GCTGAGCGTGCTGGTGACCACCTTCAGCTGGAGCAACATCACCCTGCTGCTGACCGGCCTGAACATG
    CTGGTGACCGCCCTGTACAGCCTGTACATGTTCACCACCACCCAGTGGGGCAGCCTGACCCACCACA
    TCAACAACATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCACCTGAGCCCCATCCTG
    CTGCTGAGCCTGAACCCCGACATCATCACCGGCTTCAGCAGCTAAGAGCACTGGGACGCCCACCGCC
    CCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGT
    TTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTAC
    CCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCC
    TTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGG
    GTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCC
    AGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTC
    AGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTC
    CCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCA
    CTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCC
    TTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAA
    CATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGGATACC
    TCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTACACAG
    CACGGCTTTTTCAAGGCTGTATTGAGATGGGAAAGTTAGGAAGAAGGGTGTGCTGGGCTbACCAGCCC
    ACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGATATCTCCTGAATTCAGAAA
    TTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTG
    TGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGG
    GTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAAGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCA
    GGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTC
    TCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGCACTTATCTGAAATCTTC
    CCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAGCTTCTAC
    AACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    76 OPA1-opt_ND4*-3′UTR GTGCTGCCCGCCTAGAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTGATGCTGAAGCTGATCGTGCCCACCATCATGCTGCTGCCCCTGACCTGGCTGAGCAAGAAGCACA
    TGATCTGGATCAACACCACCACCCACAGCCTGATCATCAGCATCATCCCCCTGCTGTTCTTCAACCAG
    ATCAACAACAACCTGTTCAGCTGCAGCCCCACCTTCAGCAGCGACCCCCTGACCACCCCCCTGCTGA
    TGCTGACCACCTGGCTGCTGCCCCTGACCATCATGGCCAGCCAGCGCCACCTGAGCAGCGAGCCCC
    TGAGCCGCAAGAAGCTGTACCTGAGCATGCTGATCAGCCTGCAGATCAGCCTGATCATGACCTTCACC
    GCCACCGAGCTGATCATGTTCTACATCTTCTTCGAGACCACCCTGATCCCCACCCTGGCCATCATCAC
    CCGCTGGGGCAACCAGCCCGAGCGCCTGAACGCCGGCACCTACTTCCTGTTCTACACCCTGGTGGG
    CAGCCTGCCCCTGCTGATCGCCCTGATCTACACCCACAACACCCTGGGCAGCCTGAACATCCTGCTG
    CTGACCCTGACCGCCCAGGAGCTGAGCAACAGCTGGGCCAACAACCTGATGTGGCTGGCCTACACCA
    TGGCCTTCATGGTGAAGATGCCCCTGTACGGCCTGCACCTGTGGCTGCCCAAGGCCCACGTGGAGG
    CCCCCATCGCCGGCAGCATGGTGCTGGCCGCCGTGCTGCTGAAGCTGGGCGGCTACGGCATGATGC
    GCCTGACCCTGATCCTGAACCCCCTGACCAAGCACATGGCCTACCCCTTCCTGGTGCTGAGCCTGTG
    GGGCATGATCATGACCAGCAGCATCTGCCTGCGCCAGACCGACCTGAAGAGCCTGATCGCCTACAGC
    AGCATCAGCCACATGGCCCTGGTGGTGACCGCCATCCTGATCCAGACCCCCTGGAGCTTCACCGGCG
    CCGTGATCCTGATGATCGCCCACGGCCTGACCAGCAGCCTGCTGTTCTGCCTGGCCAACAGCAACTA
    CGAGCGCACCCACAGCCGCATCATGATCCTGAGCCAGGGCCTGCAGACCCTGCTGCCCCTGATGGC
    CTTCTGGTGGCTGCTGGCCAGCCTGGCCAACCTGGCCCTGCCCCCCACCATCAACCTGCTGGGCGA
    GCTGAGCGTGCTGGTGACCACCTTCAGCTGGAGCAACATCACCCTGCTGCTGACCGGCCTGAACATG
    CTGGTGACCGCCCTGTACAGCCTGTACATGTTCACCACCACCCAGTGGGGCAGCCTGACCCACCACA
    TCAACAACATGAAGCCCAGCTTCACCCGCGAGAACACCCTGATGTTCATGCACCTGAGCCCCATCCTG
    CTGCTGAGCCTGAACCCCGACATCATCACCGGCTTCAGCAGCTAAGAGCACTGGGACGCCCACCGCC
    CCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGT
    TTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTAC
    CCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCC
    TTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGG
    GTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACATGCCC
    AGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTC
    AGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTC
    CCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCA
    CTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    77 OPA1-ND6-3′UTR GTGCTGCCCGCCTAGAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTGATGATGTATGCTTTGTTTCTGTTGAGTGTGGGTTTAGTAATGGGGTTTGTGGGGTTTTCTTCTAAG
    CCTTCTCCTATTTATGGGGGTTTAGTATTGATTGTTAGCGGTGTGGTCGGGTGTGTTATTATTCTGAAT
    TTTGGGGGAGGTTATATGGGTTTAATGGTTTTTTTAATTTATTTAGGGGGAATGATGGTTGTCTTTGGAT
    ATACTACAGCGATGGCTATTGAGGAGTATCCTGAGGCATGGGGGTCAGGGGTTGAGGTCTTGGTGAG
    TGTTTTAGTGGGGTTAGCGATGGAGGTAGGATTGGTGCTGTGGGTGAAAGAGTATGATGGGGTGGTG
    GTTGTGGTAAACTTTAATAGTGTAGGAAGCTGGATGATTTATGAAGGAGAGGGGTCAGGGTTGATTCG
    GGAGGATCCTATTGGTGCGGGGGCTTTGTATGATTATGGGCGTTGGTTAGTAGTAGTTACTGGTTGGA
    CATTGTTTGTTGGTGTATATATTGTAATTGAGATTGCTCGGGGGAATTAGGAGCACTGGGACGCCCAC
    CGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCT
    GGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAAT
    ATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTT
    TCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATG
    GGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACAT
    GCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTG
    AGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTG
    GTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTCAATACCAATAGCTAGGACCCGGCTGCT
    GTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTC
    CTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGGCATCTTTATAGTTCAC
    GTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGG
    ATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTA
    CACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGTGTGCTGGGCTAACC
    AGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTGATATCTCCTGAATTC
    AGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTT
    ACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCC
    CACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTTTTCGATTACTCAGT
    CTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAAACAACATTTAAACA
    GAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTTTGCACTTATCTGA
    AATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAATGTCTGGAAAAAG
    CTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    78 OPA1-ND6-3′UTR* GTGCTGCCCGCCTAGAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTGATGATGTATGCTTTGTTTCTGTTGAGTGTGGGTTTAGTAATGGGGTTTGTGGGGTTTTCTTCTAAG
    CCTTCTCCTATTTATGGGGGTTTAGTATTGATTGTTAGCGGTGTGGTCGGGTGTGTTATTATTCTGAAT
    TTTGGGGGAGGTTATATGGGTTTAATGGTTTTTTTAATTTATTTAGGGGGAATGATGGTTGTCTTTGGAT
    ATACTACAGCGATGGCTATTGAGGAGTATCCTGAGGCATGGGGGTCAGGGGTTGAGGTCTTGGTGAG
    TGTTTTAGTGGGGTTAGCGATGGAGGTAGGATTGGTGCTGTGGGTGAAAGAGTATGATGGGGTGGTG
    GTTGTGGTAAACTTTAATAGTGTAGGAAGCTGGATGATTTATGAAGGAGAGGGGTCAGGGTTGATTCG
    GGAGGATCCTATTGGTGCGGGGGCTTTGTATGATTATGGGCGTTGGTTAGTAGTAGTTACTGGTTGGA
    CATTGTTTGTTGGTGTATATATTGTAATTGAGATTGCTCGGGGGAATTAGGAGCACTGGGACGCCCAC
    CGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCT
    GGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAAT
    ATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACAAAAAAGGAATTATTTT
    TCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATG
    GGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCACACGCACACTCCACAT
    GCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTG
    AGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTG
    GTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGCTAGGACCCGGCTGCT
    GTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGACTGCCA
    79 OPA1-opt_ND6-3′UTR GTGCTGCCCGCCTAGAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTGATGATGTACGCCCTGTTCCTGCTGAGCGTGGGCCTGGTGATGGGCTTCGTGGGCTTCAGCAGCA
    AGCCCAGCCCCATCTACGGCGGCCTGGTGCTGATCGTGAGCGGCGTGGTGGGCTGCGTGATCATCC
    TGAACTTCGGCGGCGGCTACATGGGCCTGATGGTGTTCCTGATCTACCTGGGCGGCATGATGGTGGT
    GTTCGGCTACACCACCGCCATGGCCATCGAGGAGTACCCCGAGGCCTGGGGCAGCGGCGTGGAGGT
    GCTGGTGAGCGTGCTGGTGGGCCTGGCCATGGAGGTGGGCCTGGTGCTGTGGGTGAAGGAGTACGA
    CGGCGTGGTGGTGGTGGTGAACTTCAACAGCGTGGGCAGCTGGATGATCTACGAGGGCGAGGGCAG
    CGGCCTGATCCGCGAGGACCCCATCGGCGCCGGCGCCCTGTACGACTACGGCCGCTGGCTGGTGGT
    GGTGACCGGCTGGACCCTGTTCGTGGGCGTGTACATCGTGATCGAGATCGCCCGCGGCAACTAAGA
    GCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACA
    CAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAG
    TTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATAC
    AAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGT
    TTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCA
    CACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGC
    TGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCA
    GGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGC
    TAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGA
    CTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGG
    CATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGA
    CTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTC
    GCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGT
    GTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTG
    ATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTG
    GGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTA
    GTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTT
    TTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAA
    ACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACTGTT
    TGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAAT
    GTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    80 OPA1-opt_ND6-3′UTR′ GTGCTGCCCGCCTAGAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTGATGATGTACGCCCTGTTCCTGCTGAGCGTGGGCCTGGTGATGGGCTTCGTGGGCTTCAGCAGCA
    AGCCCAGCCCCATCTACGGCGGCCTGGTGCTGATCGTGAGCGGCGTGGTGGGCTGCGTGATCATCC
    TGAACTTCGGCGGCGGCTACATGGGCCTGATGGTGTTCCTGATCTACCTGGGCGGCATGATGGTGGT
    GTTCGGCTACACCACCGCCATGGCCATCGAGGAGTACCCCGAGGCCTGGGGCAGCGGCGTGGAGGT
    GCTGGTGAGCGTGCTGGTGGGCCTGGCCATGGAGGTGGGCCTGGTGCTGTGGGTGAAGGAGTACGA
    CGGCGTGGTGGTGGTGGTGAACTTCAACAGCGTGGGCAGCTGGATGATCTACGAGGGCGAGGGCAG
    CGGCCTGATCCGCGAGGACCCCATCGGCGCCGGCGCCCTGTACGACTACGGCCGCTGGCTGGTGGT
    GGTGACCGGCTGGACCCTGTTCGTGGGCGTGTACATCGTGATCGAGATCGCCCGCGGCAACTAAGA
    GCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACA
    CAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAG
    TTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATAC
    AAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGT
    TTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCA
    CACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGC
    TGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCA
    GGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGC
    TAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGGTGGA
    CTGCCA
    81 OPA1-ND1-3′UTR GTGCTGCCCGCCTAGAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTGATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCATTCCTAATGCTTACCGAA
    CGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGCCCCTACGGGCTACTACA
    ACCCTTCGCTGACGCCATAAAACTCTTCACCAAAGAGCCCCTAAAACCCGCCACATCTACCATCACCC
    TCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGACCCCCCTCCCCATGCCC
    AACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTAGCCTAGCCGTTTACTCA
    ATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCACTGCGAGCAGTAGCCC
    AAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTAATGAGTGGCTCCTTTAA
    CCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATCATGGCCCTTGGCCATGA
    TGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCGAAGGGGAGTCCGA
    ACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTCTTCATGGCCGAATACA
    CAAACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAACATATGACGCACTCTCCC
    CTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCTGTTCTTATGGATTCGAAC
    AGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAACTTCCTACCACTCACCC
    TAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCCCCTCAAACCTAAGAGC
    ACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACA
    AGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTT
    TTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAATACA
    AAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTT
    TCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCAC
    ACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCT
    GTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCA
    GGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGC
    TAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGA
    CTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACAGG
    CATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTGGA
    CTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGGTC
    GCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGGGT
    GTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCCTG
    ATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCTG
    GGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTTTTA
    GTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGATGTT
    TTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGATTGGTCGGGGTAGGAGAGTTAA
    ACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAAATCACTGTT
    TGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGGAAT
    GTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAGAAGCTTT
    82 OPA1-NDI-3′UTR* GTGCTGCCCGCCTAGAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTGATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCATTCCTAATGCTTACCGAA
    CGAAAAATTCTAGGCTATATGCAACTACGCAAAGGCCCCAACGTTGTAGGCCCCTACGGGCTACTACA
    ACCCTTCGCTGACGCCATAAAACTCTTCACCAAAGAGCCCCTAAAACCCGCCACATCTACCATCACCC
    TCTACATCACCGCCCCGACCTTAGCTCTCACCATCGCTCTTCTACTATGGACCCCCCTCCCCATGCCC
    AACCCCCTGGTCAACCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTAGCCTAGCCGTTTACTCA
    ATCCTCTGGTCAGGGTGGGCATCAAACTCAAACTACGCCCTGATCGGCGCACTGCGAGCAGTAGCCC
    AAACAATCTCATATGAAGTCACCCTAGCCATCATTCTACTATCAACATTACTAATGAGTGGCTCCTTTAA
    CCTCTCCACCCTTATCACAACACAAGAACACCTCTGGTTACTCCTGCCATCATGGCCCTTGGCCATGA
    TGTGGTTTATCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCGAAGGGGAGTCCGA
    ACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTCTTCATGGCCGAATACA
    CAAACATTATTATGATGAACACCCTCACCACTACAATCTTCCTAGGAACAACATATGACGCACTCTCCC
    CTGAACTCTACACAACATATTTTGTCACCAAGACCCTACTTCTAACCTCCCTGTTCTTATGGATTCGAAC
    AGCATACCCCCGATTCCGCTACGACCAACTCATGCACCTCCTATGGAAAAACTTCCTACCACTCACCC
    TAGCATTACTTATGTGGTATGTCTCCATGCCCATTACAATCTCCAGCATTCCCCCTCAAACCTAAGAGC
    ACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAACACA
    AGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGACAGTT
    TTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGGTCAGTCAGTGAATACA
    AAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTGTT
    TCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACCAC
    ACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTGCT
    GTCTCTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTTGTGACTGAGCCA
    GGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATAGC
    TAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTGGA
    CTGCCA
    83 OPA1-opt_ND1-3′UTR GTGCTGCCCGCCTAGAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTGATGGCCAACCTGCTGCTGCTGATCGTGCCCATCCTGATCGCCATGGCCTTCCTGATGCTGACCG
    AGCGCAAGATCCTGGGCTACATGCAGCTGCGCAAGGGCCCCAACGTGGTGGGCCCCTACGGCCTGC
    TGCAGCCCTTCGCCGACGCCATCAAGCTGTTCACCAAGGAGCCCCTGAAGCCCGCCACCAGCACCAT
    CACCCTGTACATCACCGCCCCCACCCTGGCCCTGACCATCGCCCTGCTGCTGTGGACCCCCCTGCCC
    ATGCCCAACCCCCTGCTGAACCTGAACCTGGGCCTGCTGTTCATCCTGGCCACCAGCAGCCTGGCCG
    TGTACAGCATCCTGTGGAGCGGCTGGGCCAGCAACAGCAACTACGCCCTGATCGGCGCCCTGCGCG
    CCGTGGCCCAGACCATCAGCTACGAGGTGACCCTGGCCATCATCCTGCTGAGCACCCTGCTGATGAG
    CGGCAGCTTCAACCTGAGCACCCTGATCACCACCCAGGAGCACCTGTGGCTGCTGCTGCCCAGCTGG
    CCCCTGGCCATGATGTGGTTCATCAGCACCCTGGCCGAGACCAACCGCACCCCCTTCGACCTGGCCG
    AGGGCGAGAGCGAGCTGGTGAGCGGCTTCAACATCGAGTACGCCGCCGGCCCCTTCGCCCTGTTCT
    TCATGGCCGAGTACACCAACATCATCATGATGAACACCCTGACCACCACCATCTTCCTGGGCACCACC
    TACGACGCCCTGAGCCCCGAGCTGTACACCACCTACTTCGTGACCAAGACCCTGCTGCTGACCAGCC
    TGTTCCTGTGGATCCGCACCGCCTACCCCCGCTTCCGCTACGACCAGCTGATGCACCTGCTGTGGAA
    GAACTTCCTGCCCCTGACCCTGGCCCTGCTGATGTGGTACGTGAGCATGCCCATCACCATCAGCAGC
    ATCCCCCCCCAGACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCAT
    GTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGT
    GCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGC
    ATCAGGTCAGTCAGTGAATACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTTATACAlCTCCTC
    CAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGT
    TCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAA
    AGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACC
    CCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGT
    CCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGC
    CTTGGGAGTCTCAAGCTGGACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCA
    GAACTCCAAGGAGTCACAGGCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCT
    TCTAAAAGGGTAGCCCTGGACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTC
    TGGAGTCACTACTGTGGGTCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAA
    GGGAAGTTAGGAAGAAGGGTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGT
    GAAAAATACATGTCCATCCTGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAA
    GAGCCAGAAGCAGGGTTCTGGGAATTTTGCAAGTTACCTGTGGCCAGGTGTGGTCTCGGTTACCAAAT
    ACGGTTACCTGCAGCTTTTTAGTCCTTTGTGCTCCCACGGGTCTACAGAGTCCCATCTGCCCAAAGGT
    CTTGAAGCTTGACAGGATGTTTTCGATTACTCAGTCTCCCAGGGCACTACTGGTCCGTAGGATTCGAT
    TGGTCGGGGTAGGAGAGTTAAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAG
    GTAGATAACATCCAATCACTGTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTAC
    TGTGGAGAACATTGCATAGGAATGTCTGGAAAAAGCTTCTACAACTTGTTACAGCCTTCACATTTGTAG
    AAGCTTT
    84 OPA1-opt_ND1-3′UTR* GTGCTGCCCGCCTAAAAGGGTGAAGTGGTTGTTTCCGTGACGGACTGAGTACGGGTGCCTGTCAGG
    CTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGG
    GCCACTTCCTGGGTCATTCCTGGACCGGGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGG
    CCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCCGCTGTGGC
    CTGATGGCCAACCTGCTGCTGCTGATCGTGCCCATCCTGATCGCCATGGCCTTCCTGATGCTGACCG
    AGCGCAAGATCCTGGGCTACATGCAGCTGCGCAAGGGCCCCAACGTGGTGGGCCCCTACGGCCTGC
    TGCAGCCCTTCGCCGACGCCATCAAGCTGTTCACCAAGGAGCCCCTGAAGCCCGCCACCAGCACCAT
    CACCCTGTACATCACCGCCCCCACCCTGGCCCTGACCATCGCCCTGCTGCTGTGGACCCCCCTGCCC
    ATGCCCAACCCCCTGGTGAACCTGAACCTGGGCCTGCTGTTCATCCTGGCCACCAGCAGCCTGGCCG
    TGTACAGCATCCTGTGGAGCGGCTGGGCCAGCAACAGCAACTACGCCCTGATCGGCGCCCTGCGCG
    CCGTGGCCCAGACCATCAGCTACGAGGTGACCCTGGCCATCATCCTGCTGAGCACCCTGCTGATGAG
    CGGCAGCTTCAACCTGAGCACCCTGATCACCACCCAGGAGCACCTGTGGCTGCTGCTGCCCAGCTGG
    CCCCTGGCCATGATGTGGTTTCATCAGCACCCTGGCCGAGACCAACCGCACCCCCTTCGACCTGGCCG
    AGGGCGAGAGCGAGCTGGTGAGCGGCTTCAACATCGAGTACGCCGCCGGCCCCTTCGCCCTGTTCT
    TCATGGCCGAGTACACCAACATCATCATGATGAACACCCTGACCACCACCATCTTCCTGGGCACCACC
    TACGACGCCCTGAGCCCCGAGCTGTACACCACCTACTTCGTGACCAAGACCCTGCTGCTGACCAGCC
    TGTTCCTGTGGATCCGCACCGCCTACCCCCGCTTCCGCTACGACCAGCTGATGCACCTGCTGTGGAA
    GAACTTCCTGCCCCTGACCCTGGCCCTGCTGATGTGGTACGTGAGCATGCCCATCACCATCAGCAGC
    ATCCCCCCCCAGACCTAAGAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCAT
    GTTGTGGTAATTCTGGAACACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGT
    GCTCAGTGATCACTTGACAGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGC
    ATCAGCTCAGTCAGTGAATACAAAAAAGGAAATTATTTTTCCCTTTGAGGGTCTTTTATACATCTCTCCTC
    CAACCCCACCCTCTATTCTGTTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGT
    TCCATCCTTACCACCACACCACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAA
    AGTGTGAGCCTCATGATCTGCTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACC
    CCCTTCCTTGTGACTGAGCCAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGT
    CCTTCTAACAATACCAATAGCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGC
    CTTGGGAGTCTCAAGCTGGACTGCCA
    85 β-actin-S primer CGAGATCGTGCGGGACAT
    86 β-actin-A primer CAGGAAGGAGGGCTGGAAC
    87 ND4-S primer CTGCCTACGACAAACAGAC
    88 ND4-A primer AGTGCGTTCGTAGTTTGAG
    89 ND6-F primer ATGATGTATGCTTTGTTTCTG
    90 ND6-R primer CTAATTCCCCCGAGCAATCTC
    91 ND6-S primer AGTGTGGGTTTAGTAATG
    92 NDS-A primer TGCCTCAGGATACTCCTC
    93 β-actin-F primer CTCCATCCTGGCCTCGCTGT
    94 β-actin-R primer GCTGTCACCTTCACCGTTCC
    95 ND6-F primer GGGTTTTCTTCTAAGCCTTCTCC
    96 ND6-R primer CCATCATACTCTTTCACCCACAG
    97 opt_ND6-F primer CGCCTGCTGACCGGCTGCGT
    98 opt_ND6-R CCAGGCCTCGGGGTACTCCT
    99 ND1-F primer ATGGCCGCATCTCCGCACACT
    100 ND1-R primer TTAGGTTTGAGGGGGAATGCT
    101 ND1-F primer AACCTCAACCTAGGCCTCCTA
    102 ND1-R primer TGGCAGGAGTAACCAGAGGTG
    103 ND1-F primer AGGAGGCTCTGTCTGGTATCTT;
    104 ND1-R primer TTTTAGGGGCTCTTTGGTGAA
    105 opt-ND1-F primer GCCGCCTGCTGACCGGCTGCGT
    106 opt-ND1-R primer TGATGTACAGGGTGATGGTGCTGG
    107 ND4-S primer GCCAACAGCAACTACGAGC
    108 ND4-A primer TGATGTTGCTCCAGCTGAAAG
    109 opt-ND4-S primer GCCTGACCCTGATCCTGAAC
    110 opt-ND4-A primer GTGCGCTCGTAGTTGCTGTT
    111 hsACO2 GGGCAGTGCCTCCCCGCCCCGCCGCTGGCGTCAAGTTCAGCTCCACGTGTGCCATCAGTGGATCCG
    ATCCGTCCAGCCATGGCTTCCTATTCCAAGATGGTGTGACCAGACATGCTTCCTGGTCCCCGCTTAGC
    CCACGGAGTGACTGTGGTTGTGGTGGGGGGGTTCTTAAAATAACTTTTTAGCCCCCGTCTTCCTATTTT
    GAGTTTGGTTCAGATCTTAAGCAGCTCCATGCAACTGTATTTATTTTTGATGACAAGACTCCCATCTAAA
    GTTTTTCTCCTGCCTGATCATTTCATTGGTGGCTGAAGGATTCTAGAGAACCTTTTGTTCTTGCAAGGA
    AAACAAGAATCCAAAACCAGTGACTGTTCTGTGA
    112 hsATP513 GGGGTCTTTGTCCTCTGTACTGTCTCTCTCCTTGCCCCTAACCCAAAAAGCTTCATTTTTCTGTGTAGG
    CTGCACAAGAGCCTTGATTGAAGATATATTCTTTCTGAACAGTATTTAAGGTTTCCAATAAAATGTACAC
    CCCTCAG
    113 hsAK2 TGTTGGGTCCAAGAAGGAATTTCTTTCCATCCCTGTGAGGCAATGGGTGGGAATGATAGGACAGGCAA
    AGAGAAGCTTCCTCAGGCTAGCAAAAATATCATTTGATGTATTGATTAAAAAAGCACTTGCTTGATGTAT
    CTTTGGCGTGTGTGCTACTCTCATCTGTGTGTATGTGTGTTGTGTGTGTGTGTGTGTGCATGCACATAT
    GTGTTCACTCTGCTACTTTGTAAGTTTTAGGCTAGGTTGCTTTACCAGCTGTTTACTTCTTTTTTGTTGTT
    GTTTTGAGACAAGGTTTCGCTCTGCCACCCTGGCTGGAGTGCAGTGGCGTGATCTTGGCTCACGGCA
    ACCTCTGCCTCCTGGGGCTCAAGCAATTATCCCACCTCAGCCTCCTGAGCAGCTGGGACTACAGGTG
    CATGCCACAACACCTGGCTGATATTTGTATTTTTTGTAGAGACAGGATTTTGCCAAGTTGCCCAGGCTG
    GTCTTGAACTCCTAGGCTTAAGCAATCCACCCACCTTGGCCTCCTGAAGTGCCAGGATCACAGACGTG
    AGCCACTACACCCAGCCCAGCTGTTTACTTCTTTAACCATACTTTTGATTTTATTTTTTGACCAAAATGA
    ACTAACCCAGGTAATCTTCCAGGGACCGCAATTCCAGAACCTCATAGTATTTCTTCCATTTCCAGCAGC
    TGATTAGAAGTCCAGGATCATGTGAAGTCAGGCAGGGTCACAGTTCCTGATGGCACATTATGGACAGA
    GAATTCCATTTTGTTTTCTAACCCATGATGAAAACCCACGTGAGTCAGTGTGTGAACAGGGATCATTAA
    TTTTTTCCCCCTAGGTGGAAGGAAAAAGGCACTTACTTTGCAGGTTACAGAAATTACTGGGAGAGGAT
    ATCGTCATAAAAAGAGCCAGGCCAAATTGGAATATTTTTGTGATCTGCATCATGATGCTGAAAATAGCA
    ATTATTTGGGAATTGGGTTTGAAAACTGAATTGTTGCCAGAGAATTAAACCAGGTGAAAGGTCCTTTTG
    AATTCAGATTGTCTTCTGAACATCCAGGCTGATCATCTGAGAGCAGTCAAATCTACTTCCCCAAAAAGA
    GACCAGGGTAGGTTTATTTGCTTTTATTTTTAATGTTTGCCTGTGTTTCCAAGTGTGAACAAAACAGTGT
    GTGATCTATTCTTGGATTCATTTTGATCAGTATTTATTCAAACCCAGTCTCTCTCCAGGACATAAAACTG
    AAATCAGATATGTTCTTTTTAAGCCCAAACCCTCTCCTTTCTAGATCCAACCCTTCACCCCTAATTTTAT
    GATGGCTATAGCCATGGACTTCCCCAAGAAAAGATCACCCAGAAATAAGACCACCTGTGACAGTTACC
    AGCTTTTATTCATAACCTTAGCTTCCCAACTATTGAGCATTTTCTAAGGTCCCTGCTGTCTTTTGGTCTC
    TGGTTTGATTTGTGGCAAACAGATGAAGTAACAGACTGCTATGAAGGACCACAAAAACGGCAGCCTCT
    GGAAAAACCATTAGAAAGTCAGTGGCAGATCCAGTAAATAATATCGCCAGCCTCAGCATAATCTGCTG
    CTGACTCGATTCAGTGGACTCTAAAGTGCCCAGCCTCCTGACCTGAGCTCTCCTGCCATCTGTGAGAC
    TACCAGAGGTCTTATCTGCTGTCCACATGGCAACTGGGCATGAGTACCTGGCCACCTTGCTTCCCTCT
    TTGCCTGGTCCAAGTGAGTGTCTGCTGCCTCTGTCCTGCCTTGTTTTCCTGGCTCTAAACCAACTCCA
    CCCACTCTTAATGGAAAACTCAGTCTGGCTTTGTGTGTTTCTGGGAAGCACATGACTTCTGGGAATGGG
    CAAGGAAGAGGAGTGAAACAAAAACTGTCAGCTATGTGTGCCTGGTCTGGGATCCTTCTCTGGGTGAC
    AGTGGCATCATGAATCTTAGAATCAGCTCCCC
    114 hsALDH2 GAATCATGCAAGCTTCCTCCCTCAGCCATTGATGGAAAGTTCAGCAAGATCAGCAACAAAACCAAGAA
    AAATGATCCTTGCGTGCTGAATATCTGAAAAGAGAAATTTTTCCTACAAAATCTCTTGGGTCAAGAAAG
    TTCTAGAATTTGAATTGATAAACATGGTGGGTTGGCTGAGGGTAAGAGTATATGAGGAACCTTTTAAAC
    GACAACAATACTGCTAGCTTTCAGGATGATTTTTAAAAAATAGATTCAAATGTGTTATCCTCTCTCTGAA
    ACGCTTCCTATAACTCGAGTTTATAGGGGAAGAAAAAGCTATTGTTTACAATTATATCACCATTAAGGCA
    ACTGCTACACCCTGCTTTGTATTCTGGGCTAAGATTCATTAAAAACTAGCTGCTCTTAACTTACA
    115 hsCOX10 GAGCACTGGGACGCCCACCGCCCCTTTCCCTCCGCTGCCAGGCGAGCATGTTGTGGTAATTCTGGAA
    CACAAGAAGAGAAATTGCTGGGTTTAGAACAAGATTATAAACGAATTCGGTGCTCAGTGATCACTTGAC
    AGTTTTTTTTTTTTTTAAATATTACCCAAAATGCTCCCCAAATAAGAAATGCATCAGCTCAGTCAGTGAAT
    ACAAAAAAGGAATTATTTTTCCCTTTGAGGGTCTTTATACATCTCTCCTCCAACCCCACCCTCTATTCTG
    TTTCTTCCTCCTCACATGGGGGTACACATACACAGCTTCCTCTTTTGGTTCCATCCTTACCACCACACC
    ACACGCACACTCCACATGCCCAGCAGAGTGGCACTTGGTGGCCAGAAAGTGTGAGCCTCATGATCTG
    CTGTCTGTAGTTCTGTGAGCTCAGGTCCCTCAAAGGCCTCGGAGCACCCCCTTCCTGGTGACTGAGC
    CAGGGCCTGCATTTTTGGTTTTCCCCACCCCACACATTCTCAACCATAGTCCTTCTAACAATACCAATA
    GCTAGGACCCGGCTGCTGTGCACTGGGACTGGGGATTCCACATGTTTGCCTTGGGAGTCTCAAGCTG
    GACTGCCAGCCCCTGTCCTCCCTTCACCCCCATTGCGTATGAGCATTTCAGAACTCCAAGGAGTCACA
    GGCATCTTTATAGTTCACGTTAACATATAGACACTGTTGGAAGCAGTTCCTTCTAAAAGGGTAGCCCTG
    GACTTAATACCAGCCGGATACCTCTGGCCCCCACCCCATTACTGTACCTCTGGAGTCACTACTGTGGG
    TCGCCACTCCTCTGCTACACAGCACGGCTTTTTCAAGGCTGTATTGAGAAGGGAAGTTAGGAAGAAGG
    GTGTGCTGGGCTAACCAGCCCACAGAGCTCACATTCCTGTCCCTTGGGTGAAAAATACATGTCCATCC
    TGATATCTCCTGAATTCAGAAATTAGCCTCCACATGTGCAATGGCTTTAAGAGCCAGAAGCAGGGTTCT
    GGGAATTTTGCAAGTTATCCTGTGGCCAGGTGTGGTCTCGGTTACCAAATACGGTTACCTGCAGCTTT
    TTAGTCCTTTGTGCTCCCACGGGTCTGCAGAGTCCCATCTGCCCAAAGGTCTTGAAGCTTGACAGGAT
    GTTTTCATTACTCAGTCTCCCAGGGCACTGCTGGTCCGTAGGGATTCATTGGTCGGGGTGGGAGAGTT
    AAACAACATTTAAACAGAGTTCTCTCAAAAATGTCTAAAGGGATTGTAGGTAGATAACATCCAATCACT
    GTTTGCACTTATCTGAAATCTTCCCTCTTGGCTGCCCCCAGGTATTTACTGTGGAGAACATTGCATAGG
    AATGTCTGGAAAAAGCCTCTACAACTTGTTACAGCCTTCACATTTGTACAATTCATTGATTCTCTTTTCC
    TTCCACAATAAAATGGTATACAAGAAC
    116 hsUQCRFS1 GAGACTTGGACTCAAGTCATAGGCTTCTTTCAGTCTTTATGTCACCTCAGGAGACTTATTTGAGAGGAA
    GCCTTCTGTACTTGAAGTTGATTTGAAATATGTAAAGAAATTGATGATGTATTTGCAAACATTAATGTGAAA
    TAAATTGAATTTAATGTTGAATACTTTCAGGCATTCACTTAATAAAGACACTGTTAAGCACTGTTATGCT
    CAGTCATACACGCGAAAGGTACAATGTCTTTTAGCTAATTCTAATTAAAAATTACAGACTGGTGTACAA
    GATACTTGTG
    117 hsNDUFV1 CCCACCACCCTGGCCTGCTGTCCTGCGTCTATCCATGTGGAATGCTGGACAATAAAGCGAGTGCTGC
    CCACCCTCCAGCTGCC
    118 hsNDUFV2 TTTATATTGAACTGTAAATATGTCACTAGAGAAATAAAATATGGACTTCCAATCTACGTAAACTTA
    119 hsSOD2 ACCACGATCGTTATGCTGAGTATGTTAAGCTCTTTATGACTGTTTTTGTAGTGGTATAGAGTACTGCAG
    AATACAGTAAGCTGCTCTATTGTAGCATTTCTTGATGTTGCTTAGTCACTTATTTCATAAACAACTTAAT
    GTTCTGAATAATTTCTTACTAAACATTTTGTTATTGGGCAAGTGATTGAAAATAGTAAATGCTTTGTGTG
    ATTGA
    120 hsCOX6c TCTTGGAATATAAAGAATTTCTTCAGGTTGAATTACCTAGAAGTTTGTCACTGACTTGTGTTCCTGAACT
    ATGACACATGAATATGTGGGCTAAGAAATAGTTCCTCTTGATAAATAAACAATTAACAAATACTTTGGAC
    AGTAAGTCTTTCTCAGTTCTTAATGATAATGCAGGGCACTTACTAGCATAAGAATTGGTTTGGGATTTAA
    CTGTTTATGAAGCTAACTTGATTTCCGTGTTTTGTTAAAATTTCATTGTTCTAGCACATCTTTAACTGTGA
    TAGTT
    121 hsIRP1 GAGACGTGCACTTGGTCGTGCGCCCAGGGAGGAAGCCGCACCACCAGCCAGCGCAGGCCCTGGTG
    GAGAGGCCTCCCTGGCTGCCTCTGGGAGGGGTGCTGCCTTGTAGATGGAGCAAGTGAGCACTGAGG
    GTCTGGTGCCAATCCTGTAGGCACAAAACCAGAAGTTTCTACATTCTCTATTTTTGTTAATCATCTTCTC
    TTTTTCCAGAATTTGGAAGCTAGAATGGTGGGAATGTCAGTAGTGCCAGAAAGAGAGAACCAAGCTTG
    TCTTTAAAGTTACTGATCACAGGACGTTGCTTTTTCACTGTTTCCTATTAATCTTCAGCTGAACACAAGC
    AAACCTTCTCAGGAGGTGTCTCCTACCCTCTTATTGTTCCTCTTACGCTCTGCTCAATGAAACCTTCCT
    CTTGAGGGTCATTTTCCTTTCTGTATTAATTATACCAGTGTTAAGTGACATAGATAAGAACTTTGCACAC
    TTCAAATCAGAGCAGTGATTCTCTCTTCTCTCCCCTTTTCCTTCAGAGTGAATCATCCAGACTCCTCAT
    GGATAGGTCGGGTGTTAAAGTTGTTTTGATTATGTACCTTTTGATAGATCCACATAAAAAGAAATGTGA
    AGTTTTCTTTTACTATCTTTTCATTTATCAAGCAGAGACCTTTGTTGGGAGGCGGTTTGGGAGAACACA
    TTTCTAATTTGAATGAAATGAAATCTATTTTCAGTG
    122 hsMRPS12 CAGAAGAAGTGACGGCTGGGGGCACAGTGGGCTGGGCGCCCCTGCAGAACATGAACCTTCCGCTCC
    TGGCTGCCACAGGGTCCTCCGATGCTGGCCTTTGCGCCTCTAGAGGCAGCCACTCATGGATTCAAGT
    CCTGGCTCCGCCTCTTCCATCAGGACCACT
    123 hsATP5J2 AGAGGACACACTCTGCACCCCCCCACCCCACGACCTTGGCCCGAGCCCCTCCGTGAGGAA
    124 mSOD2 AGCCCTTCCGCCAGGCTGTGTGTCAGGCCCGTGGTGGGTGTTTTGTAGTAGTGTAGAGCATTGCA
    125 hsCXA1L CTTATGTTCTGTGCGCATTCTGGCAGGAATTCTGTCTCTTCAGAGACTCATCCTCAAAACAAGACTTGA
    CACTGTGTCCTTGCCCCAGTCCTAGGAACTGTGGCACACAGAGATGTTCATTTTAAAAACGGATTTCAT
    GAAACACTCTTGTACTTATGTTTATAAGAGAGCACTGGGTAGCCAAGTGATCTTCCCATTCACAGAGTT
    AGTAAACCTCTGTACTACATGCTG
    126 MTS-COX10 MAASPHTLSSRLLTGCVGGSVWYLERRT
    127 MTS-COX8 MSVLTRLLLRGLTRLGSAAPVRRARIHSL
    128 MTS-OPA1 MWRLRRAAVA
    129 hsCOX10 MAASPHTLSSRLLTGCVGGSVWYLERRT
    130 scRPM2 MAFKSHYSKGYHRSAAQKKTATSPFDSSYQYLRQNQGLVNSDPVLHASHLHPHPVVVANVNYNNVDDILH
    PHDLDSSINNTNNPLTHEELLYNQNVSLRSLKQQQSTNYVNNNNNNQHRYY
    131 IcSirt5 MRKRSLRCHLWSANASLSPRKDEVTSRKESENLVKGKKNKKSHLHLLTFTASKTGTDSVFDVQKSKECCKE
    LGLLFTSLIHSIGSFPFDEEPKAAAVFLPGSLPQLTVLVLAPGSGSCPTGKSTPHLAASGRNAELLRPQNSMI
    VRQFTCRGTISSHLCAHLRKPRDSRNMARP
    132 tbNDUS7 MLRRTSFNFTGRAMISRGSPEWSHRLDLKKGKKTTMMHKLGTSKPNNALQYAQMTL
    133 ncQCR2 MISRSALSRGSQLALRRPAAAKTACRGFAAAAASPAASYEPTTIAG
    134 hsATP5G2 MPELILYVAITLSVAERLVGPGHACAEPSFRSSRCSAPLCLLCSGSSSPATAPHPLKMFACSKFVSTPSLVK
    STSQLLSRPLSAVVLKRPEILTDESLSSLAVSCPLTSLVSSRSFQTSAISRDIDTA
    135 hsLACTB MYRLMSAVTARAAAPGGLASSCGRRGVHCRAGLPPLGHGWVGGLGLGLGLALGVKLAGGLRGAAPAQS
    PAAPDPEASPLAEPPQEQSLAPWSPCTPAPPCSRCFARAIESSRDLL
    136 spilv1 MTVLAPLRRLHTRAAFSSYGREIALQKRPLNLNSCSAVRRYGTGFSNNLRIKKLKNAFGVVRANSTKSTSTV
    TTASPIKYDSSFVGKTGGEEIFHDMMLKHNVKFTVFGYPGGAILPVFDAIYRSPHFEFILPRHEQAAGHA
    137 gmCOX2 MILCPLEAFTVQHILTISVMGLLSCFRSTVLRKCSKGSSGMSRFLYTNNFQRNLISSGGNESYYGYFNRRSY
    TSLYMGTGTVGGITSARIRVPNVGCEGFMCSSHLSITQRNSRLIHSTSKIVPN
    138 crATP6 MALQQAAPRVFGLLGRAPVALGQSGILTGSSGFKNQGFNGSLQSVENHVYAQAFSTSSQEEQAAPSIQGA
    SGMKLPGMAGSMLLGKSRSGLRTGSMVPFAAQQAMNM
    139 hsOPA1 MWRLRRAAVACEVCQSLVKHSSGTKGSLPLQKLHLVSRSIYHSHHPTLKLQRPQLRTSFQQFSSLTNLPLR
    KLKFSPIKYGYQPRRN
    140 hsSDHD MAVLWRLSAVCGALGGRALLLRTPVVRPAHTSAFLQDRPTPEWCGVQHIHLSPSHH
    141 hsADCK3 MAAILGDTIMVAKGLVKLTQAAVETHLQHLGIGGELIMAARALQSTAVEQIGMFLGKVQGQDKHEEYFAENF
    GGPEGEFHPSVPHAAGASTDFSSASAPDQSAPPSLGHAHSEGPAPAYVASGPFREAGFPGQASSPLGRA
    NGRLFANPRDSFSAMGFQRRF
    142 osP0644B0624-2 MALLLRHSPKLRRAHAILGCERGTVVRHFSSSTCSSLVKEDTVSSSNLHPEYAKKIGGSDFSHDRQSGKEL
    QNFKVSPQEASRASNFMRASKYGMPITANGVHSLFSCGQVVPSRCF
    143 Neurospora crassa ATP9 (ncATP9) MASTRVLASRLASQMAASAKVARPAVRVAQVSKRTIQTGSPLQTLKRTQMTSIVNATTRQAFQKRA
    144 hsGHITM MLAARLVCLRTLPSRVFHPAFTKASPVVKNSITKNQWLLTPSRE
    145 hsNDUFAB1 MASRVLSAYVSRLPAAFAPLPRVRMLAVARPLSTALCSAGTQTRLGTLQPALVLAQVPGRVTQLCRQY
    146 hsATP5G3 MFACAKLACTPSLIRAGSRVAYRPISASVLSRPEASRTGEGSTVFNGAQNGVSQLICREFQTSAISR
    147 crATP6_hsADCK3 MALQQAAPRVFGLLGRAPVALGQSGILTGSSGFKNQGFNGSLQSVENHVYAQAFSTSSQEEQAAPSIQGA
    SGMKLPGMAGSMLLGKSRSGLRTGSMVPFAAQQAMNMCGMAAILGDTIMVAKGLVKLTQAAVETHLQHL
    GIGGELIMAARALQSTAVEQIGMFLGKVQGQDKHEEYFAENFGGPEGEFHFSVPHAAGASTDFSSASAPD
    QSAPPSLGHAHSEGPAPAYVASGPFREAGFPGQASSPLGRANGRLFANPRDSFSAMGFQRRFGG
    148 ncATP9_ncATP9 MASTRVLASRLASCMAASAKVARPAVRVAQVSKRTIQTGSPLQTLKRTQMTSIVNATTRQAFQKRAMAST
    RVLASRLASQMAASAKVARPAVRVAQVSKRTIQTGSPLQTLKRTQMTSIVNATTRQAFQKRA
    149 zmLOC100282174 MALLRAAVSELRRRGRGALTPLPALSSLLSSLSPRSPASTRPEPNNPHADRRHVIALRRCPPLPASAVLAPE
    LLHARGLLPRHWSHASPLSTSSSSSRPADKAQLTWVDKWIPEAARPY
    150 ncATP9_zmLOC100282174_spilv1_ncATP9 MASTRVLASRLASQMAASAKVARPAVRVAQVSKRTIQTGSPLQTLKRTQMTSIVNATTRQAFQKRAMALLR
    AAVSELRRRGRGALTPLPALSSLLSSLSPRSPASTRPEPNNPHADRRHVIALRRCPPLPASAVLAPELLHAR
    GLLPRHWSHASPLSTSSSSSRPADKAQLTWVDKWIPEAARPYMTVLAPLRRLHTRAAFSSYGREIALQKRF
    LNLNSCSAVRRYGTGFSNNLRIKKLKNAFGVVRANSTKSTSTVTTASPIKYDSSFVGKTGGEIFHDMMLKH
    NVKHVFGYPGGAILPVFDAIYRSPHFEFILPRHEQAAGHAMASTRVLASRLASQMAASAKVARPAVRVAQV
    SKRTIQTGSPLQTLKRTQMTSIVNATTRQAFQKRA
    151 zmLOC100282174_hsADCK3_crATP6_hsATP5G3 MALLRAAVSELRRRGRGALTPLPALSSLTSSLSPRSPASTRPERNNPHADRRHVIALRRCPPLPASAVLAPE
    LLHARGLLPRHWSHASPLSTSSSSSRPADKAQLTWVDKWIPEAARPYMAAILGDTIMVAKGLVKLTQAAVE
    THLQHLGIGGELIMAARALQSTAVEQIGMFLGKVQGQDKHEEYFAENFGGPEGEFHFSVPHAAGASTDFS
    SASAPDQSAPPSLGHAHSEGPAPAYVASGPFREAGFPGQASSPLGRANGRLFANPRDSFSAMGFQRRF
    MALQQAAPRVFGLTGRAPVALGQSGILTGSSGFKNQGFNGSLQSVENHVYAQAFSTSSQEEQAAPSIQGA
    SGMKLPGMAGSMLLGKSRSGLRTGSMVPFAAQQAMNMMFACAKLACTPSLIRAGSRVAYRPISASVLSR
    PEASRTGEGSTVFNGAQNGVSQLIQREFQTSAISR
    152 zmLOC100282174_hsADCK3_hsATP5G3 MALLRAAVSELRRRGRGALTPLPALSSLLSSLSPRSPASTRPEPNNPHADRRHVIALRRCPPLPASAVLAPE
    LLHARGLLPRHWSHASPLSTSSSSSRPADKAQLTWVDKWIPEAARPYMAAILGDTIMVAKGLVKLTQAAVE
    THLQHLGIGGELIMAARALQSTAVEQIGMFLGKVQGQDKHEEYFAENFGGPEGEFHFSVPHAAGASTDFS
    SASAPDCSAPPSLGHAHSEGPAPAYVASGPFREAGFPGQASSPLGRANGRLFANPRDSFSAMGFQRRF
    MFACAKLACTPSLIRAGSRVAYRPISASVLSRPEASRTGEGSTVFNGAQNGVSQLIQREFQTSAISR
    153 ncATP9_zmLOC100282174 MASTRVLASRLASQMAASAKVARPAVRVAQVSKRTIQTGSPLQTLKRTQMTSIVNATTRQAFQKRAMALLR
    AAVSELRRRGRGALTPLPALSSLLSSLSPRSPASTRPEPNNPHADRRHVIALRRCPPLPASAVLAPELLHAR
    GLLPRHWSHASPLSTSSSSSRPADKAQLTWVDKWIPEAARPY
    154 hsADCK3_zmLOC100262174_crATP6_hsATP5G3 MAAILGDTIMVAKGLVKLTQAAVETHLQFTLGIGGELIMAARALQSTAVEQIGMFLGKVQGQDKHEEYFAENF
    GGPEGEFHFSVPHAAGASTDFSSASAPDQSAPPSLGHAHSEGPAPAYVASGPFREAGFPGQASSPLGRA
    NGRLFANPRDSFSAMGFQRRFMALLRAAVSELRRRGRGALTPLPALSSLLSSLSPRSPASTRPERNNPHA
    DRRHVIALRRCPPLPASAVLAPELLHARGLLPRHWSHASPLSTSSSSSRPADKAQLTWVDKWIPEAARPY
    MALQQAAPRVFGLLGRARVALGQSGILTGSSGFKNQGFNGSLQSVENHVYAQAFSTSSQEEQAAPSIQGA
    SGMKLPGMAGSMLLGKSRSGLRTGSMVPFAAQQAMNMMFACAKLACTPSLIRAGSRVAYRPISASVLSR
    PEASRTGEGSTVFNGAQNGVSQLIQREFQTSAISR
    155 crATP6_hsADCK3_zmLOC100282174_hsATP5G3 MALQQAAPRVFGLLGRAPVALGQSGILTGSSGFKNQGFNGSLQSVENHVYAQAFSTSSQEEQAAPSIQGA
    SGMKLPGMAGSMLLGKSRSGLRTGSMVPFAAQQAMNMMAAILGDTIMVAKGLVKLTQAAVETHLQHLGIG
    GELIMAARALQSTAVEQIGMFLGKVQGQDKHEEYFAENFGGPEGEFHFSVPHAAGASTDFSSASAPDQSA
    PPSLGHANSEGPAPAYVASGPFREAGFPGQASSPLGRANGRLFANPRDSFSAMGFQRRFMALLRAAVSE
    LRRRGRGALTPLPALSSLLSSLSPRSPASTRPEPNNPHADRRHVIALRRCPPLPASAVLAPELLHARGLLPR
    HWSHASPLSTSSSSSRPADKAQLTWVDKWIPEAARPYMFACAKLACTPSLIRAGSRVAYRPISASVLSRPE
    ASRTGEGSTVFNGAQNGVSQLIQREFQTSAISR
    156 hsADCK3_zmLOC100282174 MAAILGDTIMVAKGLVKLTQAAVETHLQHLGIGGELIMAARALQSTAVEQIGMFLGKVQGQDKHEEYFAENF
    GGPEGEFHFSVPHAAGASTDFSSASAPDQSAPPSLGHAHSEGPAPAYVASGPFREAGFPGQASSPLGRA
    NGRLFANPRDSFSAMGFQRRFGGMALLRAAVSELRRPGRGALTPLPALSSLLSSLSPRSPASTRPEPNNP
    HADRRHVIALRRCPPLPASAVLAPELLHARGLLPRHWSHASPLSTSSSSSRPADKAQLTWVDKWIPEAARP
    YGG
    157 hsADCK_zmLOC100282174_crATP6 MAAILGDTIMVAKGLVKLTQAAVETHLQFTLGIGGELIMAARALQSTAVEQIGMFLGKVQGQDKHEEYFAENF
    GGPEGEFHFSVPHAAGASTDFSSASAPDQSAPPSLGHAHSEGPAPAYVASGPFREAGFPGQASSPLGRA
    NGRLFANPRDSFSAMGFQRRFGGMALLRAAVSELRRRGRGALTPLPALSSLLSSLSPRSPASTRPEPNNP
    HADRRHVIALRRCPPLPASAVLAPELLHARGLLPRHWSHASPLSTSSSSSRPADKACLTWVDKWIPEAARP
    YGGMALQQAAPRVFGLLGRAPVALGQSGILTGSSGFKNQGFNGSLQSVENHVYAQAFSTSSQEEQAAPSI
    QGASGMKLPGMAGSMLLGKSRSGLRTGSMVPFAAQQAMNMGG
    158 ncATP9_zmLOC100282174_spilv1_GNFP_ncATP9 MASTRVLASRLASQMAASAKVARPAVRVAQVSKRTIQTGSPLQTLKRTQMTSIVNATTRQAFQKRAMALLR
    AAVSELRRRGRGALTPLPALSSLLSSLSPRSPASTRPEPNNPHADRRHVIALRRCPPLPASAVLAPELLHAR
    GLLPRHWSHASPLSTSSSSSRPADKAQLTWVDKWIPEAARPYMTVLARLRRLHTRAAFSSYGRETALQKRF
    LNLNSCSAVRRYGTGFSNNLRIKKLKNAFGVVRANSTKSTSTVTTASPIKYDSSFVGKTGGEIFHDMMLKH
    NVKHVFGYPGGAILPVFDAIYRSPHFEFILPRHEQAAGHAVSGEGDATYGKLTLKFICTTGKLPVPWPTLVT
    TLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKED
    GNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQS
    ALSKDPNEMASTRVLASRLASQMAASAKVARPAVRVAQVSKRTIQTGSPLQTLKRTQMTSIVNARQAFQ
    KRA
    159 ncATP9_zmLOC10028217_spilv1_IcSirt5_osP0644B06.24- MASTRVLASRLASCMAASAKVARPAVRVAQVSKRTIQTGSPLQTLKRTQMTSIVNATTRQAFQKRAMALLR
    2_hsATP5G2_ncATP9 AAVSELRRRGRGALTPLPALSSLLSSLSPRSPASTRPEPNNPHADRRHVIALRRCPPLPASAVLAPELLHAR
    GLLPRHWSHASPLSTSSSSSRPADKAQLTWVDKWIPEAARPYMTVLAPLRRLHTRAAFSSYGREIALQKRF
    LNLNSCSAVRRYGTGFSNNLRIKKLKNAFGVVRANSTKSTSTVTTASPIKYDSSFVGKTGGEIFHDMMLKH
    NVKHVFGYPGGAILPVFDAIYRSPHFEFILPRHEQAAGHAMRKRSLRCHLWSANASLSPRKDEVTSRKESE
    NLVKGKKNKKSHLHLLLFTASKIGTDSVFDVQKSKECCKELGLLFTSLIHSIGSFPFDEEPKAAAVFLPGSLP
    QLTVLVLAPGSGSCPTGKSTPHLAASGRNAELLRPQNSMIVRQFTCRGTISSHLCAHLRKPHDSRNMARP
    MALLLRHSPKLRRAHAILGCERGTVVRHFSSSTCSSLVKEDTVSSSNLHPEYAKKIGGSDFSHDRQSGKEL
    QNFKVSPQEASRASNFMRASKYGMPITANGVHSLFSCGQVVPSRCFMPELILYVAITLSVAERLVGPGHAC
    AEPSFRSSRCSAPLCLLCSGSSSPATAPHPLKMFACSKFVSTPSLVKSTSQLLSRPLSAVVLKRPEILTDES
    LSSLAVSCPLTSLVSSRSFQTSAISRDIDTAMASTRVLASRLASQMAASAKVARPAVRVAQVSKRTIQTGSP
    LQTLKRTQMTSIVNATTRQAFQKRA
    160 ND4 MLKLIVPTIMLLPLTWLSKKHMIWINTTTHSLIISIIPLLFFNQTNNNLFSCSPTFSSDPLTTPLLMLTTWLLPLTI
    MASQRHLSSEPLSRKKLYLSMLISLQISLIMTFTATELIMFYIFFETTLIPTLAIITRWGNQPERLNAGTYFLFYT
    LVGSLPLLIALTHNTLGSLNILLLTLTAQELSNSWANNLMWLAYTMAFMVKMPLYGLHLWPKAHVEAPIA
    GSMVLAAVLLKLGGYGMMRLTLILNPLTKHMAYPFLVSLWGMIMTSSICLRQTDLKSLIAYSSISHMALVVT
    AILIQTPWSFTGAVILMIAHGLTSSLLFCLANSNYERTHSRIMILSQGLQTLLPLMAFWWLLASLANLALPPTIN
    LLGELSVLVTTFSWSNITLLLTGLNMLVTALYSLYMFTTTQWGSLTHHINNMKPSFTRENTLMFMHLSPILLL
    SLNPDIITGFSS
    161 ND6 MMYALFLLSVGLVMGFVGFSSKPSPIYGGLVLIVSGVVGCVIILNFGGGYMGLMVFLIYLGGMMVVFGYTTA
    MAIEEYPEAWGSGVEVLVSVLVGLAMEVGLVLVWKEYDGVVVVVNFNSVGSWMIYEGEGSGLIREDPIGA
    GALYDYGRWLVVVTGWTLFVGVYIVIEIARGN
    162 ND1 MANLLLLIVPILIAMAFLMLTERKILGYMQLRKGPNVVGPYGLLQPFADAIKLFTKEPLKPATSTITLYITAPTLA
    LTIALLLWTPLPMPNPLVNLNLGLLFILATSSLAVYSILWSGWASNSNYALIGALRAVAQTISYEVTLAIILLSTL
    LMSGSFNLSTLITTQEHLWLLLPSWPLAMMWFISTLAETNRTPFDLAEGESELVSGFNIEYAAGPFALFFMA
    EYTNIIMMNTLTTTIFLGTTYDALSPELYTTYFVTKTLLLTSLFLWIRTAYPRFRYDQLMHLLWKNFLPLTLALL
    MWYVSMPITISSIPPQT
    • Adeno-Associated Virus (AAV)
  • Adeno-associated virus (AAV) is a small virus that infects humans and some other primate species. The compositions disclosed herein comprises firstly an adeno-associated virus (AAV) genome or a derivative thereof.
  • An AAV genome is a polynucleotide sequence which encodes functions needed for production of an AAV viral particle. These functions include those operating in the replication and packaging cycle for AAV in a host cell, including encapsidation of the AAV genome into an AAV viral particle. Naturally occurring AAV viruses are replication-deficient and rely on the provision of helper functions in trans for completion of a replication and packaging cycle. Accordingly, the AAV genome of the vector of the invention is typically replication-deficient.
  • The AAV genome can be in single-stranded form, either positive or negative-sense, or alternatively in double-stranded form. The use of a double-stranded form allows bypass of the DNA replication step in the target cell and so can accelerate transgene expression.
  • The AAV genome may be from any naturally derived serotype or isolate or Glade of AAV. Thus, the AAV genome may be the full genome of a naturally occurring AAV virus. As is known to the skilled person, AAV viruses occurring in nature may be classified according to various biological systems.
  • Commonly, AAV viruses are referred to in terms of their serotype. A serotype corresponds to a variant subspecies of AAV which owing to its profile of expression of capsid surface antigens has a distinctive reactivity which can be used to distinguish it from other variant subspecies. Typically, a virus having a particular AAV serotype does not efficiently cross-react with neutralising antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, also recombinant serotypes, such as Rec2 and Rec3, recently identified from primate brain.
  • A preferred serotype of AAV for use in the invention is AAV2. Other serotypes of particular interest for use in the invention include AAV4, AAV5 and AAV8 which efficiently transduce tissue in the eye, such as the retinal pigmented epithelium. The serotype of AAV which is used can be an AAV serotype which is not AAV4. Reviews of AAV serotypes may be found in Choi et al (Curr Gene Ther. 2005; 5(3); 299-310) and Wu et al (Molecular Therapy. 2006; 14(3), 316-327). The sequences of AAV genomes or of elements of AAV genomes including ITR sequences, rep or cap genes for use in the invention may be derived from the following accession numbers for AAV whole genome sequences: Adeno-associated virus 1 NC_002077, AF063497; Adeno-associated virus 2 NC_001401; Adeno-associated virus 3 NC_001729; Adeno-associated virus 3B NC_001863; Adeno-associated virus 4 NC_001829; Adeno-associated virus 5 Y18065, AF085716; Adeno-associated virus 6 NC_001862; Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; Avian AAV strain DA-1 NC 006263, AY629583; Bovine AAV NC_005889, AY388617.
  • AAV viruses may also be referred to in terms of clades or clones. This refers to the phylogenetic relationship of naturally derived AAV viruses, and typically to a phylogenetic group of AAV viruses which can be traced back to a common ancestor, and includes all descendants thereof. Additionally, AAV viruses may be referred to in terms of a specific isolate, i.e. a genetic isolate of a specific AAV virus found in nature. The term genetic isolate describes a population of AAV viruses which has undergone limited genetic mixing with other naturally occurring AAV viruses, thereby defining a recognisably distinct population at a genetic level.
  • Examples of clades and isolates of AAV that may be used in the invention include: Clade A: AAV1 NC_002077, AF063497, AAV6 NC_001862, Hu. 48 AY530611, Hu 43 AY530606, Hu 44 AY530607, Hu 46 AY530609; Clade B: Hu. 19 AY530584, Hu. 20 AY530586, Hu 23 AY530589, Hu22 AY530588, Hu24 AY530590, Hu21 AY530587, Hu27 AY530592, Hu28 AY530593, Hu 29 AY530594, Hu63 AY530624, Hu64 AY530625, Hu13 AY530578, Hu56 AY530618, Hu57 AY530619, Hu49 AY530612, Hu58 AY530620, Hu34 AY530598, Hu35 AY530599, AAV2 NC_001401. Hu45 AY530608, Hu47 AY530610, Hu5I AY530613, Hu52 AY530614, Hu T41 AY695378, Hu S17 AY695376, Hu T88 AY695375, Hu T71 AY695374, Hu T70 AY695373, Hu T40 AY695372, Hu T32 AY695371, Hu T17 AY695370, Hu LG15 AY695377; Clade C: Hu9 AY530629, Hu10 AY530576, Hul1 AY530577, Hu53 AY530615, Hu55 AY530617, Hu54 AY530616, Hu7 AY530628, Hu18 AY530583, Hu15 AY530580, Hu16 AY530581, Hu25 AY530591, Hu60 AY530622, Ch5 AY243021, Hu3 AY530595, Hu1 AY530575, Hu4 AY530602 Hu2, AY530585, Hu61 AY530623; Clade D: Rh62 AY530573, Rh48 AY530561, Rh54 AY530567, Rh55 AY530568, Cy2 AY243020, AAV7 AF513851, Rh35 AY243000, Rh37 AY242998, Rh36 AY242999, Cy6 AY243016, Cy4 AY243018, Cy3 AY243019, Cy5 AY243017, Rh13 AY243013; Clade E: Rh38 AY530558, Hu66 AY530626, Hu42 AY530605, Hu67 AY530627, Hu40 AY530603, Hu41 AY530604, Hu37 AY530600, Rh40 AY530559, Rh2 AY243007, Bb1 AY243023, Bb2 AY243022, Rh10 AY243015, Hu17 AY530582, Hu6 AY530621, Rh25 AY530557, Pi2 AY530554, Pi1 AY530553, Pi3 AY530555, Rh57 AY530569, Rh50 AY530563, Rh49 AY530562, Hu39 AY530601, Rh58 AY530570, Rh61 AY530572, Rh52 AY530565, Rh53 AY530566, Rh51 AY530564, Rh64 AY530574, Rh43 AY530560, AAV8 AF513852, Rh8 AY242997, Rh1 AY530556; Clade F: Hu14 (AAV9) AY530579, Hu31 AY530596, Hu32 AY530597, Clonal Isolate AAV5 Y18065, AF085716, AAV 3 NC_001729, AAV 3B NC_001863, AAV4 NC_001829, Rh34 AY243001, Rh33 AY243002, Rh32 AY243003.
  • The skilled person can select an appropriate serotype, Glade, clone or isolate of AAV for use in the present invention on the basis of their common general knowledge. For instance, the AAV5 capsid has been shown to transduce primate cone photoreceptors efficiently as evidenced by the successful correction of an inherited color vision defect (Mancuso et al., Nature 2009, 461:784-7).
  • It should be understood however that the invention also encompasses use of an AAV genome of other serotypes that may not yet have been identified or characterised. The AAV serotype determines the tissue specificity of infection (or tropism) of an AAV virus. Accordingly, preferred AAV serotypes for use in AAV viruses administered to patients in accordance with the invention are those which have natural tropism for or a high efficiency of infection of target cells within eye in LHON. Thus, AAV serotypes for use in AAV viruses administered to patients can be ones which infect cells of the neurosensory retina and retinal pigment epithelium.
  • Typically, the AAV genome of a naturally derived serotype or isolate or Glade of AAV comprises at least one inverted terminal repeat sequence (ITR). An ITR sequence acts in cis to provide a functional origin of replication, and allows for integration and excision of the vector from the genome of a cell. In preferred embodiments, one or more ITR sequences flank the polynucleotide sequence encoding ND4, ND6, or ND1 or a variant thereof. Preferred ITR sequences are those of AAV2, and variants thereof. The AAV genome typically also comprises packaging genes, such as rep and/or cap genes which encode packaging functions for an AAV viral particle. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof. The cap gene encodes one or more capsid proteins such as VP1, VP2 and VP3 or variants thereof. These proteins make up the capsid of an AAV viral particle. Capsid variants are discussed below.
  • A promoter will be operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19 and p40 promoters (Laughlin et al., 1979, PNAS, 76:5567-5571). For example, the p5 and p19 promoters are generally used to express the rep gene, while the p40 promoter is generally used to express the cap gene.
  • As discussed above, the AAV genome used in the vector of the invention may therefore be the full genome of a naturally occurring AAV virus. For example, a vector comprising a full AAV genome may be used to prepare AAV virus in vitro. However, while such a vector may in principle be administered to patients, this will be done rarely in practice. Preferably the AAV genome will be derivatised for the purpose of administration to patients. Such derivatisation is standard in the art and the present invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art. Derivatisation of the AAV genome and of the AAV capsid are reviewed in Coura and Nardi (Virology Journal, 2007, 4:99), and in Choi et al and Wu et al, referenced above.
  • Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a ND4, ND6, or ND1 transgene from a vector of the invention in vivo. Typically, it is possible to truncate the AAV genome significantly to include minimal viral sequence yet retain the above function. This is preferred for safety reasons to reduce the risk of recombination of the vector with wild-type virus, and also to avoid triggering a cellular immune response by the presence of viral gene proteins in the target cell.
  • Typically, a derivative will include at least one inverted terminal repeat sequence (ITR), preferably more than one ITR, such as two ITRs or more. One or more of the ITRs may be derived from AAV genomes having different serotypes, or may be a chimeric or mutant ITR. A preferred mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication of the genome to generate a single-stranded genome which contains both coding and complementary sequences i.e. a self-complementary AAV genome. This allows for bypass of DNA replication in the target cell, and so enables accelerated transgene expression.
  • The one or more ITRs will preferably flank the polynucleotide sequence encoding ND4, ND6, ND1, or a variant thereof at either end. The inclusion of one or more ITRs is preferred to aid concatamer formation of the vector of the invention in the nucleus of a host cell, for example following the conversion of single-stranded vector DNA into double-stranded DNA by the action of host cell DNA polymerases. The formation of such episomal concatamers protects the vector construct during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo.
  • In preferred embodiments, ITR elements will be the only sequences retained from the native AAV genome in the derivative. Thus, a derivative will preferably not include the rep and/or cap genes of the native genome and any other sequences of the native genome. This is preferred for the reasons described above, and also to reduce the possibility of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the transgene.
  • With reference to the AAV2 genome, the following portions could therefore be removed in a derivative of the invention: One inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes (NB: the rep gene in the wildtype AAV genome should not to be confused with ND4, ND6, or ND1, the human gene affected in LHON). However, in some embodiments, including in vitro embodiments, derivatives may additionally include one or more rep and/or cap genes or other viral sequences of an AAV genome. Naturally occurring AAV virus integrates with a high frequency at a specific site on human chromosome 19, and shows a negligible frequency of random integration, such that retention of an integrative capacity in the vector may be tolerated in a therapeutic setting.
  • Where a derivative genome comprises genes encoding capsid proteins i.e. VP1. VP2 and/or VP3, the derivative may be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAV viruses. In particular, the invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones, or isolates of AAV within the same vector i.e. pseudotyping.
  • Chimeric, shuffled or capsid-modified derivatives will be typically selected to provide one or more desired functionalities for the viral vector. Thus, these derivatives may display increased efficiency of gene delivery, decreased immunogenicity (humoral or cellular), an altered tropism range and/or improved targeting of a particular cell type compared to an AAV viral vector comprising a naturally occurring AAV genome, such as that of AAV2. Increased efficiency of gene delivery may be effected by improved receptor or co-receptor binding at the cell surface, improved internalisation, improved trafficking within the cell and into the nucleus, improved uncoating of the viral particle and improved conversion of a single-stranded genome to double-stranded form. Increased efficiency may also relate to an altered tropism range or targeting of a specific cell population, such that the vector dose is not diluted by administration to tissues where it is not needed.
  • Chimeric capsid proteins include those generated by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This may be performed for example by a marker rescue approach in which non-infectious capsid sequences of one serotype are cotransfected with capsid sequences of a different serotype, and directed selection is used to select for capsid sequences having desired properties. The capsid sequences of the different serotypes can be altered by homologous recombination within the cell to produce novel chimeric capsid proteins.
  • Chimeric capsid proteins also include those generated by engineering of capsid protein sequences to transfer specific capsid protein domains, surface loops or specific amino acid residues between two or more capsid proteins, for example between two or more capsid proteins of different serotypes.
  • Shuffled or chimeric capsid proteins may also be generated by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be created by randomly fragmenting the sequences of related AAV genes e.g. those encoding capsid proteins of multiple different serotypes and then subsequently reassembling the fragments in a self-priming polymerase reaction, which may also cause crossovers in regions of sequence homology. A library of hybrid AAV genes created in this way by shuffling the capsid genes of several serotypes can be screened to identify viral clones having a desired functionality. Similarly, error prone PCR may be used to randomly mutate AAV capsid genes to create a diverse library of variants which may then be selected for a desired property.
  • The sequences of the capsid genes may also be genetically modified to introduce specific deletions, substitutions or insertions with respect to the native wild-type sequence. In particular, capsid genes may be modified by the insertion of a sequence of an unrelated protein or peptide within an open reading frame of a capsid coding sequence, or at the N- and/or C-terminus of a capsid coding sequence.
  • The unrelated protein or peptide may advantageously be one which acts as a ligand for a particular cell type, thereby conferring improved binding to a target cell or improving the specificity of targeting of the vector to a particular cell population. An example might include the use of RGD peptide to block uptake in the retinal pigment epithelium and thereby enhance transduction of surrounding retinal tissues (Cronin et al., 2008 ARVO Abstract: D1048). The unrelated protein may also be one which assists purification of the viral particle as part of the production process i.e. an epitope or affinity tag. The site of insertion will typically be selected so as not to interfere with other functions of the viral particle e.g. internalisation, trafficking of the viral particle. The skilled person can identify suitable sites for insertion based on their common general knowledge. Particular sites are disclosed in Choi et al, referenced above.
  • The invention additionally encompasses the provision of sequences of an AAV genome in a different order and configuration to that of a native AAV genome. The invention also encompasses the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus. Such chimeric genes may be composed of sequences from two or more related viral proteins of different viral species.
  • The vector of the invention takes the form of a polynucleotide sequence comprising an AAV genome or derivative thereof and a sequence encoding ND4, ND6, ND1 or a variant thereof.
  • For the avoidance of doubt, the invention also provides an AAV viral particle comprising a vector of the invention. The AAV particles of the invention include transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype. The AAV particles of the invention also include mosaic forms wherein a mixture of unmodified capsid proteins from two or more different serotypes makes up the viral envelope The AAV particle also includes chemically modified forms bearing ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting a particular cell surface receptor.
  • The invention additionally provides a host cell comprising a vector or AAV viral particle of the invention.
    • Recombinant Nucleic Acid Sequences
  • Also disclosed herein are recombinant nucleic acid sequences comprising a polynucleotide sequence encoding a NADH dehydrogenase subunit-4 (ND4), NADH dehydrogenase subunit-1 (ND1) and NADH dehydrogenase subunit-6 (ND6) polypeptide or a variant thereof.
  • The polynucleotide sequence for ND4 is shown in SEQ ID NO: 6 and encodes the protein shown in SEQ ID NO: 160. Further nucleic acid sequences for ND4 are SEQ ID NO: 7 and 8. The polynucleotide sequence for ND6 is shown in SEQ ID NO: 9 and encodes the protein shown in SEQ ID NO: 161. A further nucleic acid sequence for ND6 is SEQ ID NO: 10. The polynucleotide sequence for ND1 is shown in SEQ ID NO: 11 and encodes the protein shown in SEQ ID NO: 162. A further nucleic acid sequence for ND1 is SEQ ID NO: 12.
  • A variant of any one of SEQ ID NO: 160, 161, or 162 may comprise truncations, mutants or homologues thereof, and any transcript variants thereof which encode a functional ND4, ND6, or ND1 polypeptide. Any homologues mentioned herein are typically at least 70% homologous to a relevant region of ND4, ND6, or ND1, and can functionally compensate for the polypeptide deficiency.
  • Homology can be measured using known methods. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et at (1984) Nucleic Acids Research 12, 387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et at (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • In preferred embodiments, a recombinant nucleic acid sequence may encode a polypeptide which is at least 55%, 65%, 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 97%, 99%, 99.5%, or 100% homologous to a relevant region of ND4, ND6, or ND1 (SEQ ID NO: 160, 161, or 162) over at least 20, preferably at least 30, for instance at least 40, 60, 100, 200, 300, 400 or more contiguous amino acids, or even over the entire sequence of the recombinant nucleic acid. The relevant region will be one which provides for functional activity of ND4, ND6, or ND1.
  • Alternatively, and preferably the recombinant nucleic acid sequence may encode a polypeptide having at least 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 97%, 99%, 99.5%, or 100% homologous to full-length ND4, ND6, or ND1 (SEQ ID NO: 160, 161, or 162) over its entire sequence. Typically the recombinant nucleic acid sequence differs from the relevant region of ND4, ND6, or ND1 (SEQ ID NO: 160, 161, or 162) by at least, or less than, 2, 5, 10, 20, 40, 50 or 60 mutations (each of which can be substitutions, insertions or deletions).
  • A recombinant nucleic acid ND4, ND6, or ND1 polypeptide may have a percentage identity with a particular region of SEQ ID NO: 160, 161, or 162 which is the same as any of the specific percentage homology values (i.e. it may have at least 70%, 80% or 90% and more preferably at least 95%, 97/o, 99% identity) across any of the lengths of sequence mentioned above.
  • Variants of ND4, ND6, or ND1 (SEQ ID NO: 160, 161, or 162) also include truncations. Any truncation may be used so long as the variant is still functional. Truncations will typically be made to remove sequences that are non-essential for the protein activity and/or do not affect conformation of the folded protein, in particular folding of the active site. Appropriate truncations can routinely be identified by systematic truncation of sequences of varying length from the N- or C-terminus. Preferred truncations are N-terminal and may remove all other sequences except for the catalytic domain.
  • Variants of ND4, ND6, or ND1 (SEQ ID NO: 160, 161, or 162) further include mutants which have one or more, for example, 2, 3, 4, 5 to 10, 10 to 20, 20 to 40 or more, amino acid insertions, substitutions or deletions with respect to a particular region of ND4, ND6, or ND1 (SEQ ID NO: 160, 161, or 162). Deletions and insertions are made preferably outside of the catalytic domain as described below. Substitutions are also typically made in regions that are non-essential for protease activity and/or do not affect conformation of the folded protein.
  • Substitutions preferably introduce one or more conservative changes, which replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative change may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and may be selected in accordance with the properties of the amino acids.
  • Similarly, preferred variants of the polynucleotide sequence of ND4. ND6, or ND1 (SEQ ID NO: 6, 9, or 11) include polynucleotides having at least 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 96%, 97%, 98%, 99%, or 99.5% homologous to a relevant region of ND4, ND6, or ND1 (SEQ ID NO: 6, 9, or 11). Preferably the variant displays these levels of homology to full-length ND4, ND6, or ND1 (SEQ ID NO: 6, 9, or 11) over its entire sequence.
  • Mitochondrial targeting sequences (MTSs) and three prime untranslated regions (3′UTRs) can be used to target proteins or mRNA to the mitochondria. The charge, length, and structure of the MTS can be important for protein import into the mitochondria. Particular 3′UTRs may drive mRNA localization to the mitochondrial surface and thus facilitate cotranslational protein import into the mitochondria.
  • The polynucleotide sequence for a mitochondrial targeting sequence can encode a polypeptide selected from hsCOX10, hsCOX8, scRPM2, lcSirt5, tbNDUS7, ncQCR2, hsATP5G2, hsLACTB, spilv1, gmCOX2, crATP6, hsOPA1, hsSDHD, hsADCK3, osP0644B06.24-2, Neurospora crassa ATP9 (ncATP9), hsGHITM, hsNDUFAB1, hsATP5G3, crATP6_hsADCK3, ncATP9_ncATP9, zmLOC100282174, ncATP9_zmLOC100282174_spilv1_ncATP9, zmLOCI00282174_hsADCK3_crATP6_hsATP5G3, zmLOC100282174_hsADCK3_hsATP5G3, ncATP9_zmLOC100282174, hsADCK3_zmLOC100282174_crATP6_hsATP5G3, crATP6_hsADCK3_zmLOC100282174_hsATP5G3, hsADCK3_zmLOC100282174, hsADCK3_zmLOC100282174_crATP6, ncATP9_zmLOC100282174_spily_GNFP_ncATP9, and ncATP9_zmLOC100282174_spilv1_lcSirt5_osP0644B06.24-2_hsATP5G2_ncATP9 (see Table 1 for SEQ ID NO). In one example, the polynucleotide sequences, COX10 (SEQ ID NO: 1, 2, or 3) can encode the mitochondrial targeting sequence, MTS-COX10 (SEQ ID NO: 126). In another example, the polynucleotide sequences, COX8 (SEQ ID NO: 4) can encode the mitochondrial targeting sequence, MTS-COX8 (SEQ ID NO: 127). In another example, the polynucleotide sequences, OPA1 (SEQ ID NO: 5) can encode the mitochondrial targeting sequence, MTS-OPA 1 (SEQ ID NO: 128).
  • The 3′UTR nucleic acid sequence can be selected from hsACO2 (SEQ ID NO: 111), hsATP5B (SEQ ID NO: 112), hsAK2 (SEQ ID NO: 113), hsALDH2 (SEQ ID NO: 114), hsCOXI0 (SEQ ID NO: 115), hsUQCRFS1 (SEQ ID NO: 116), hsNDUFV1 (SEQ ID NO: 117), hsNDUFV2 (SEQ ID NO: 118), hsSOD2 (SEQ ID NO: 119), hsCOX6c (SEQ ID NO: 120), hsIRPl (SEQ ID NO: 121), hsMRPS12 (SEQ ID NO: 122), hsATP5J2 (SEQ ID NO: 123), mSOD2 (SEQ ID NO: 124), and hsOXA1L (SEQ ID NO: 125). The 3′UTR nucleic acid sequence can also be a variant having at least 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% homologous to any 3′UTR nucleic acid sequence listed here. For example, the 3′UTR nucleic acid sequence can be SEQ ID NO: 13 or 14.
  • Also disclosed herein are recombinant nucleic acid sequences comprising a mitochondrial targeting sequence, a mitochondrial protein coding sequence, and a 3′UTR nucleic acid sequence. For example, the recombinant nucleic acid sequence can be selected from SEQ ID NO: 15-84. The recombinant nucleic acid sequence can also be a variant having at least 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% homologous to any recombinant nucleic acid sequence listed here.
    • Promoters and Regulatory Sequences
  • The vector of the invention also includes elements allowing for the expression of the disclosed transgene in vitro or in vivo. Thus, the vector typically comprises a promoter sequence operably linked to the polynucleotide sequence encoding the ND4, ND6, or ND1 transgene or a variant thereof.
  • Any suitable promoter may be used. The promoter sequence may be constitutively active i.e. operational in any host cell background, or alternatively may be active only in a specific host cell environment, thus allowing for targeted expression of the transgene in a particular cell type. The promoter may show inducible expression in response to presence of another factor, for example a factor present in a host cell. In any event, where the vector is administered for therapy, the promoter must be functional in a retinal cell background.
  • In some embodiments, it is preferred that the promoter shows retinal-cell specific expression in order to allow for the transgene to only be expressed in retinal cell populations. Thus, expression from the promoter may be retinal-cell specific, for example confined only to cells of the neurosensory retina and retinal pigment epithelium.
  • Preferred promoters for the ND4, ND6, or ND1 transgene include the chicken beta-actin (CBA) promoter, optionally in combination with a cytomegalovirus (CME) enhancer element. In some cases, the preferred promoters for the ND4, ND6, or ND1 transgene comprises the CAG promoter. A particularly preferred promoter is a hybrid CBA/CAG promoter, for example the promoter used in the rAVE expression cassette. Examples of promoters based on human sequences that would induce retina specific gene expression include rhodospin kinase for rods and cones (Allocca et al., 2007, J Viol 81:11372-80), PR2.1 for cones only (Mancuso et al. 2009. Nature) and/or RPE65 for the retinal pigment epithelium (Bainbridge et al., 2008, N Eng J Med).
  • The vector of the invention may also comprise one or more additional regulatory sequences with may act pre- or post-transcriptionally. The regulatory sequence may be part of the native ND4, ND6, or ND1 gene locus or may be a heterologous regulatory sequence. The vector of the invention may comprise portions of the 5′UTR or 3′UTR from the native ND4, ND6, or ND1 transcript.
  • Regulatory sequences are any sequences which facilitate expression of the transgene i.e. act to increase expression of a transcript, improve nuclear export of mRNA or enhance its stability. Such regulatory sequences include for example enhancer elements, postregulatory elements and polyadenylation sites. A preferred polyadenylation site is the Bovine Growth Hormone poly-A signal. In the context of the vector of the invention such regulatory sequences will be cis-acting. However, the invention also encompasses the use of trans-acting regulatory sequences located on additional genetic constructs.
  • A preferred postregulatory element for use in a vector of the invention is the woodchuck hepatitis postregulatory element (WPRE) or a variant thereof. Another regulatory sequence which may be used in a vector of the present invention is a scaffold-attachment region (SAR). Additional regulatory sequences may be selected by the skilled person on the basis of their common general knowledge.
    • Preparation of Vector
  • The vector of the invention may be prepared by standard means known in the art for provision of vectors for gene therapy. Thus, well established public domain transfection, packaging and purification methods can be used to prepare a suitable vector preparation.
  • As discussed above, a vector of the invention may comprise the full genome of a naturally occurring AAV virus in addition to a polynucleotide sequence encoding ND4, ND6, or ND1 or a variant thereof. However, commonly a derivatised genome will be used, for instance a derivative which has at least one inverted terminal repeat sequence (ITR), but which may lack any AAV genes such as rcp or cap.
  • In such embodiments, in order to provide for assembly of the derivatised genome into an AAV viral particle, additional genetic constructs providing AAV and/or helper virus functions will be provided in a host cell in combination with the derivatised genome. These additional constructs will typically contain genes encoding structural AAV capsid proteins i.e. cap, VP1. VP2, VP3, and genes encoding other functions required for the AAV life cycle, such as rep. The selection of structural capsid proteins provided on the additional construct will determine the serotype of the packaged viral vector.
  • A particularly preferred packaged viral vector for use in the invention comprises a derivatised genome of AAV2 in combination with AAV5 or AAV8 capsid proteins. This packaged viral vector typically comprises one or more AAV2 ITRs.
  • As mentioned above, AAV viruses are replication incompetent and so helper virus functions, preferably adenovirus helper functions will typically also be provided on one or more additional constructs to allow for AAV replication.
  • All of the above additional constructs may be provided as plasmids or other episomal elements in the host cell, or alternatively one or more constructs may be integrated into the genome of the host cell.
  • In these aspects, the invention provides a method for production of a vector of the invention. The method comprises providing a vector which comprises an adeno-associated virus (AAV) genome or a derivative thereof and a polynucleotide sequence encoding ND4, ND6, or ND1 or a variant thereof in a host cell, and providing means for replication and assembly of the vector into an AAV viral particle. Preferably, the method comprises providing a vector comprising a derivative of an AAV genome and a polynucleotide sequence encoding ND4, ND6, or ND1 or a variant thereof, together with one or more additional genetic constructs encoding AAV and/or helper virus functions. Typically, the derivative of an AAV genome comprises at least one ITR. Optionally, the method further comprises a step of purifying the assembled viral particles. Additionally, the method may comprise a step of formulating the viral particles for therapeutic use.
    • Methods of Therapy and Medical Uses
  • As discussed above, the present inventors have surprisingly demonstrated that a vector of the invention may be used to address the cellular dysfunction underlying LHON. In particular, they have shown that use of the vector can correct the defect associated with LHON. This provides a means whereby the degenerative process of the disease can be treated, arrested, palliated or prevented.
  • The invention therefore provides a method of treating or preventing LHON in a patient in need thereof, comprising administering a therapeutically effective amount of a vector of the invention to the patient by direct retinal, subretinal or intravitreal injection. Accordingly, LHON is thereby treated or prevented in the patient.
  • In a related aspect, the invention provides for use of a vector of the invention in a method of treating or preventing LHON by administering said vector to a patient by direct retinal, subretinal or intravitreal injection. Additionally, the invention provides the use of a vector of the invention in the manufacture of a medicament for treating or preventing LHON by direct retinal, subretinal or intravitreal injection.
  • In all these embodiments, the vector of the invention may be administered in order to prevent the onset of one or more symptoms of LHON. The patient may be asymptomatic. The subject may have a predisposition to the disease. The method or use may comprise a step of identifying whether or not a subject is at risk of developing, or has, LHON. A prophylactically effective amount of the vector is administered to such a subject. A prophylactically effective amount is an amount which prevents the onset of one or more symptoms of the disease.
  • Alternatively, the vector may be administered once the symptoms of the disease have appeared in a subject i.e. to cure existing symptoms of the disease. A therapeutically effective amount of the antagonist is administered to such a subject. A therapeutically effective amount is an amount which is effective to ameliorate one or more symptoms of the disease. Such an amount may also arrest, slow or reverse some loss of peripheral vision associated with LHON. Such an amount may also arrest, slow or reverse onset of LHON.
  • A typical single dose is between 1010 and 1012 genome particles, depending on the amount of remaining retinal tissue that requires transduction. A genome particle is defined herein as an AAV capsid that contains a single stranded DNA molecule that can be quantified with a sequence specific method (such as real-time PCR). That dose may be provided as a single dose, but may be repeated for the fellow eye or in cases where vector may not have targeted the correct region of retina for whatever reason (such as surgical complication). The treatment is preferably a single permanent treatment for each eye, but repeat injections, for example in future years and/or with different AAV serotypes may be considered.
  • The invention also provides a method of monitoring treatment or prevention of LHON in a patient comprising measuring activity ex vivo in retinal cells obtained from said patient following administration of the AAV vector of the invention by direct retinal, subretinal or intravitreal injection. This method can allow for determination of the efficacy of treatment.
    • Pharmaceutical Compositions
  • The vector of the invention can be formulated into pharmaceutical compositions. These compositions may comprise, in addition to the vector, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may be determined by the skilled person according to the route of administration, i.e. here direct retinal, subretinal or intravitreal injection.
  • The pharmaceutical composition is typically in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, a surfactant, such as pluronic acid (PF68) 0.001% may be used.
  • For injection at the site of affliction, the active ingredient will be in the form of an aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • For delayed release, the vector may be included in a pharmaceutical composition which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.
    • Samples
  • Samples that are suitable for use in the methods described herein can be nucleic acid samples from a subject. A “nucleic acid sample” as used herein can include RNA or DNA, or a combination thereof. In another embodiment, a “polypeptide sample” (e.g., peptides or proteins, or fragments therefrom) can be used to ascertain information that an amino acid change has occurred, which is the result of a genetic variant. Nucleic acids and polypeptides can be extracted from one or more samples including but not limited to, blood, saliva, urine, mucosal scrapings of the lining of the mouth, expectorant, serum, tears, skin, tissue, or hair. A nucleic acid sample can be assayed for nucleic acid information. “Nucleic acid information,” as used herein, includes a nucleic acid sequence itself, the presence/absence of genetic variation in the nucleic acid sequence, a physical property which varies depending on the nucleic acid sequence (e.g., Tm), and the amount of the nucleic acid (e.g., number of mRNA copies). A “nucleic acid” means any one of DNA, RNA, DNA including artificial nucleotides, or RNA including artificial nucleotides. As used herein, a “purified nucleic acid” includes cDNAs, fragments of genomic nucleic acids, nucleic acids produced using the polymerase chain reaction (PCR), nucleic acids formed by restriction enzyme treatment of genomic nucleic acids, recombinant nucleic acids, and chemically synthesized nucleic acid molecules. A “recombinant” nucleic acid molecule includes a nucleic acid molecule made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. As used herein, a “polypeptide” includes proteins, fragments of proteins, and peptides, whether isolated from natural sources, produced by recombinant techniques, or chemically synthesized. A polypeptide may have one or more modifications, such as a post-translational modification (e.g., glycosylation, phosphorylation, etc.) or any other modification (e.g., pegylation, etc.). The polypeptide may contain one or more non-naturally-occurring amino acids (e.g., such as an amino acid with a side chain modification).
  • In some embodiments, the nucleic acid sample can comprise cells or tissue, for example, cell lines. Exemplary cell types from which nucleic acids can be obtained using the methods described herein include, but are not limited to, the following: a blood cell such as a B lymphocyte, T lymphocyte, leukocyte, erythrocyte, macrophage, or neutrophil; a muscle cell such as a skeletal cell, smooth muscle cell or cardiac muscle cell; a germ cell, such as a sperm or egg; an epithelial cell; a connective tissue cell, such as an adipocyte, chondrocyte; fibroblast or osteoblast; a neuron; an astrocyte; a stromal cell; an organ specific cell, such as a kidney cell, pancreatic cell, liver cell, or a keratinocyte; a stem cell; or any cell that develops therefrom. A cell from which nucleic acids can be obtained can be a blood cell or a particular type of blood cell including, for example, a hematopoietic stem cell or a cell that arises from a hematopoietic stem cell such as a red blood cell, B lymphocyte, T lymphocyte, natural killer cell, neutrophil, basophil, eosinophil, monocyte, macrophage, or platelet. Generally, any type of stem cell can be used including, without limitation, an embryonic stem cell, adult stem cell, or pluripotent stem cell.
  • In some embodiments, a nucleic acid sample can be processed for RNA or DNA isolation, for example, RNA or DNA in a cell or tissue sample can be separated from other components of the nucleic acid sample. Cells can be harvested from a nucleic acid sample using standard techniques, for example, by centrifuging a cell sample and resuspending the pelleted cells, for example, in a buffered solution, for example, phosphate-buffered saline (PBS). In some embodiments, after centrifuging the cell suspension to obtain a cell pellet, the cells can be lysed to extract DNA. In some embodiments, the nucleic acid sample can be concentrated and/or purified to isolate DNA. All nucleic acid samples obtained from a subject, including those subjected to any sort of further processing, are considered to be obtained from the subject. In some embodiments, standard techniques and kits known in the art can be used to extract RNA or DNA from a nucleic acid sample, including, for example, phenol extraction, a QIAAMP® Tissue Kit (Qiagen, Chatsworth, Calif.), a WIZARD® Genomic DNA purification kit (Promega), or a Qiagen Autopure method using Puregene chemistry, which can enable purification of highly stable DNA well-suited for archiving.
  • In some embodiments, determining the identity of an allele or determining copy number can, but need not, include obtaining a nucleic acid sample comprising RNA and/or DNA from a subject, and/or assessing the identity, copy number, presence or absence of one or more genetic variations and their chromosomal locations within the genomic DNA (i.e. subject's genome) derived from the nucleic acid sample.
  • The individual or organization that performs the determination need not actually carry out the physical analysis of a nucleic acid sample from a subject. In some embodiments, the methods can include using information obtained by analysis of the nucleic acid sample by a third party. In some embodiments, the methods can include steps that occur at more than one site. For example, a nucleic acid sample can be obtained from a subject at a first site, such as at a health care provider or at the subject's home in the case of a self-testing kit. The nucleic acid sample can be analyzed at the same or a second site, for example, at a laboratory or other testing facility.
    • Nucleic Acids
  • The nucleic acids and polypeptides described herein can be used in methods and kits of the present disclosure. In some embodiments, aptamers that specifically bind the nucleic acids and polypeptides described herein can be used in methods and kits of the present disclosure. As used herein, a nucleic acid can comprise a deoxyribonucleotide (DNA) or ribonucleotide (RNA), whether singular or in polymers, naturally occurring or non-naturally occurring, double-stranded or single-stranded, coding, for example a translated gene, or non-coding, for example a regulatory region, or any fragments, derivatives, mimetics or complements thereof. In some embodiments, nucleic acids can comprise oligonucleotides, nucleotides, polynucleotides, nucleic acid sequences, genomic sequences, complementary DNA (cDNA), antisense nucleic acids, DNA regions, probes, primers, genes, regulatory regions, introns, exons, open-reading frames, binding sites, target nucleic acids and allele-specific nucleic acids.
  • A “probe,” as used herein, includes a nucleic acid fragment for examining a nucleic acid in a specimen using the hybridization reaction based on the complementarity of nucleic acid.
  • A “hybrid” as used herein, includes a double strand formed between any one of the abovementioned nucleic acid, within the same type, or across different types, including DNA-DNA, DNA-RNA, RNA-RNA or the like.
  • “Isolated” nucleic acids, as used herein, are separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library). For example, isolated nucleic acids of the disclosure can be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material can form part of a composition, for example, a crude extract containing other substances, buffer system or reagent mix. In some embodiments, the material can be purified to essential homogeneity using methods known in the art, for example, by polyacrylamide gel electrophoresis (PAGE) or column chromatography (e.g., HPLC). With regard to genomic DNA (gDNA), the term “isolated” also can refer to nucleic acids that are separated from the chromosome with which the genomic DNA is naturally associated. For example, the isolated nucleic acid molecule can contain less than about 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotides that flank the nucleic acid molecule in the gDNA of the cell from which the nucleic acid molecule is derived.
  • Nucleic acids can be fused to other coding or regulatory sequences can be considered isolated. For example, recombinant DNA contained in a vector is included in the definition of “isolated” as used herein. In some embodiments, isolated nucleic acids can include recombinant DNA molecules in heterologous host cells or heterologous organisms, as well as partially or substantially purified DNA molecules in solution. Isolated nucleic acids also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present disclosure. An isolated nucleic acid molecule or nucleotide sequence can be synthesized chemically or by recombinant means. Such isolated nucleotide sequences can be useful, for example, in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene, in tissue (e.g., human tissue), such as by Northern blot analysis or other hybridization techniques disclosed herein. The disclosure also pertains to nucleic acid sequences that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein Such nucleic acid sequences can be detected and/or isolated by allele- or sequence-specific hybridization (e.g., under high stringency conditions). Stringency conditions and methods for nucleic acid hybridizations are well known to the skilled person (see, e.g., Current Protocols in Molecular Biology, Ausubel. F. et al., John Wiley & Sons, (1998), and Kraus, M. and Aaronson. S., Methods Enzymol., 200:546-556 (1991), the entire teachings of which are incorporated by reference herein.
  • Calculations of “identity” or “percent identity” between two or more nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The nucleotides at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e. % identity=# of identical positions/total # of positions×100). For example, a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • In some embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, of the length of the reference sequence. The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A non-limiting example of such a mathematical algorithm is described in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90-5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0), as described in Altschul, S. et al., Nucleic Acids Res., 25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, any relevant parameters of the respective programs (e.g., NBLAST) can be used. For example, parameters for sequence comparison can be set at score=100, word length=12, or can be varied (e.g., W=5 or W=20). Other examples include the algorithm of Myers and Miller, CABIOS (1989). ADVANCE, ADAM, BLAT, and FASTA. In some embodiments, the percent identity between two amino acid sequences can be accomplished using, for example, the GAP program in the GCG software package (Accelrys, Cambridge, UK).
  • “Probes” or “primers” can be oligonucleotides that hybridize in a base-specific manner to a complementary strand of a nucleic acid molecule. Probes can include primers, which can be a single-stranded oligonucleotide probe that can act as a point of initiation of template-directed DNA synthesis using methods including but not limited to, polymerase chain reaction (PCR) and ligase chain reaction (LCR) for amplification of a target sequence. Oligonucleotides, as described herein, can include segments or fragments of nucleic acid sequences, or their complements. In some embodiments, DNA segments can be between 5 and 10,000 contiguous bases, and can range from 5, 10, 12, 15, 20, or 25 nucleotides to 10, 15, 20, 25, 30, 40, 50, 100, 200, 500, 1000 or 10,000 nucleotides. In addition to DNA and RNA, probes and primers can include polypeptide nucleic acids (PNA), as described in Nielsen, P. et al., Science 254: 1497-1500 (1991). A probe or primer can comprise a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and in certain embodiments about 40, 50, 60 or 75, consecutive nucleotides of a nucleic acid molecule.
  • The present disclosure also provides isolated nucleic acids, for example, probes or primers, that contain a fragment or portion that can selectively hybridize to a nucleic acid that comprises, or consists of, a nucleotide sequence, wherein the nucleotide sequence can comprise at least one polymorphism or polymorphic allele contained in the genetic variations described herein or the wild-type nucleotide that is located at the same position, or the complements thereof. In some embodiments, the probe or primer can be at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical, to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence.
  • In some embodiments, a nucleic acid probe can be an oligonucleotide capable of hybridizing with a complementary region of a gene associated with a condition (e.g., LHON) containing a genetic variation described herein. The nucleic acid fragments of the disclosure can be used as probes or primers in assays such as those described herein.
  • The nucleic acids of the disclosure, such as those described above, can be identified and isolated using standard molecular biology techniques well known to the skilled person. In some embodiments, DNA can be amplified and/or can be labeled (e.g., radiolabeled, fluorescently labeled) and used as a probe for screening, for example, a cDNA library derived from an organism. cDNA can be derived from mRNA and can be contained in a suitable vector. For example, corresponding clones can be isolated, DNA obtained fallowing in vivo excision, and the cloned insert can be sequenced in either or both orientations by art-recognized methods to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight. Using these or similar methods, the polypeptide and the DNA encoding the polypeptide can be isolated, sequenced and further characterized.
  • In some embodiments, nucleic acid can comprise one or more polymorphisms, variations, or mutations, for example, single nucleotide polymorphisms (SNPs), single nucleotide variations (SNVs), copy number variations (CNVs), for example, insertions, deletions, inversions, and translocations. In some embodiments, nucleic acids can comprise analogs, for example, phosphorothioates, phosphoramidates, methyl phosphonate, chiralmethyl phosphonates, 2-O-methyl ribonucleotides, or modified nucleic acids, for example, modified backbone residues or linkages, or nucleic acids combined with carbohydrates, lipids, polypeptide or other materials, or peptide nucleic acids (PNAs), for example, chromatin, ribosomes, and transcriptosomes. In some embodiments nucleic acids can comprise nucleic acids in various structures, for example, A DNA, B DNA, Z-form DNA, siRNA, tRNA, and ribozymes. In some embodiments, the nucleic acid may be naturally or non-naturally polymorphic, for example, having one or more sequence differences, for example, additions, deletions and/or substitutions, as compared to a reference sequence. In some embodiments, a reference sequence can be based on publicly available information, for example, the U.C. Santa Cruz Human Genome Browser Gateway (genome.ucsc.edu/cgi-bin/hgGateway) or the NCBI website (www.ncbi.nlm.nih.gov). In some embodiments, a reference sequence can be determined by a practitioner of the present disclosure using methods well known in the art, for example, by sequencing a reference nucleic acid.
  • In some embodiments, a probe can hybridize to an allele, SNP, SNV, or CNV as described herein. In some embodiments, the probe can bind to another marker sequence associated with LHON as described herein.
  • One of skill in the art would know how to design a probe so that sequence specific hybridization can occur only if a particular allele is present in a genomic sequence from a test nucleic acid sample. The disclosure can also be reduced to practice using any convenient genotyping method, including commercially available technologies and methods for genotyping particular genetic variations
  • Control probes can also be used, for example, a probe that binds a less variable sequence, for example, a repetitive DNA associated with a centromere of a chromosome, can be used as a control. In some embodiments, probes can be obtained from commercial sources. In some embodiments, probes can be synthesized, for example, chemically or in vitro, or made from chromosomal or genomic DNA through standard techniques. In some embodiments sources of DNA that can be used include genomic DNA, cloned DNA sequences, somatic cell hybrids that contain one, or a part of one, human chromosome along with the normal chromosome complement of the host, and chromosomes purified by flow cytometry or microdissection. The region of interest can be isolated through cloning, or by site-specific amplification using PCR.
  • One or more nucleic acids for example, a probe or primer, can also be labeled, for example, by direct labeling, to comprise a detectable label. A detectable label can comprise any label capable of detection by a physical, chemical, or a biological process for example, a radioactive label, such as 32P or 3H, a fluorescent label, such as FITC, a chromophore label, an afinity-ligand label, an enzyme label, such as alkaline phosphatase, horseradish peroxidase, or 12 galactosidase, an enzyme cofactor label, a hapten conjugate label, such as digoxigenin or dinitrophenyl, a Raman signal generating label, a magnetic label, a spin label, an epitope label, such as the FLAG or HA epitope, a luminescent label, a heavy atom label, a nanoparticle label, an electrochemical label, a light scattering label, a spherical shell label, semiconductor nanocrystal label, such as quantum dots (described in U.S. Pat. No. 6,207,392), and probes labeled with any other signal generating label known to those of skill in the art, wherein a label can allow the probe to be visualized with or without a secondary detection molecule. A nucleotide can be directly incorporated into a probe with standard techniques, for example, nick translation, random priming, and PCR labeling. A “signal,” as used herein, include a signal suitably detectable and measurable by appropriate means, including fluorescence, radioactivity, chemiluminescence, and the like.
  • Non-limiting examples of label moieties useful for detection include, without limitation, suitable enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; members of a binding pair that are capable of forming complexes such as streptavidin/biotin, avidin/biotin or an antigen/antibody complex including, for example, rabbit IgG and anti-rabbit IgG; fluorophores such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, tetramethyl rhodamine, cosin, green fluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue, Texas Red, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, fluorescent lanthanide complexes such as those including Europium and Terbium, cyanine dye family members, such as Cy3 and Cy5, molecular beacons and fluorescent derivatives thereof, as well as others known in the art as described, for example, in Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999) and the 6th Edition of the Molecular Probes Handbook by Richard P. Hoagland; a luminescent material such as luminol; light scattering or plasmon resonant materials such as gold or silver particles or quantum dots; or radioactive material include 14C, 123I, 124I, 125I, Tc99m, 32P, 33P, 35S or 3H.
  • I Other labels can also be used in the methods of the present disclosure, for example, backbone labels. Backbone labels comprise nucleic acid stains that bind nucleic acids in a sequence independent manner. Non-limiting examples include intercalating dyes such as phenanthridines and acridines (e.g., ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidium monoazide, and ACMA); some minor grove binders such as indoles and imidazoles (e.g., Hoechst 33258, Hoechst 33342, Hoechst 34580 and DAPI); and miscellaneous nucleic acid stains such as acridine orange (also capable of intercalating), 7-AAD, actinomycin D, LDS751, and hydroxystilbamidine. All of the aforementioned nucleic acid stains are commercially available from suppliers such as Molecular Probes, Inc. Still other examples of nucleic acid stains include the following dyes from Molecular Probes: cyanine dyes such as SYTOX Blue, SYTOX Green, SYTOX Orange. POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red).
  • In some embodiments, fluorophores of different colors can be chosen, for example, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), 5-(and-6)-carboxy-X-rhodamine, lissamine rhodamine B, 5-(and-6)-carboxyfluorescein, fluoreseein-5-isothiocyanate (FITC), 7-diethylaminocoumarin-3-carboxvlic acid, tetramethvlrhodamine-5-(and-6)-isothiocvanate, 5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylic acid, 6-[fluorescein 5-(and-6)-carboxamido]hexanoic acid, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionic acid, cosin-5-isothiocyanate, erythrosin-5-isothiocyanate, TRITC, rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas Red, Phar-Red, allophycocvanin (APC), and CASCADE™ blue acetylazide, such that each probe in or not in a set can be distinctly visualized. In some embodiments, fluorescently labeled probes can be viewed with a fluorescence microscope and an appropriate filter for each fluorophore, or by using dual or triple band-pass filter sets to observe multiple fluorophores. In some embodiments, techniques such as flow cytometry can be used to examine the hybridization pattern of the probes.
  • In other embodiments, the probes can be indirectly labeled, for example, with biotin or digoxygenin, or labeled with radioactive isotopes such as 32P and/or 3H. As a non-limiting example, a probe indirectly labeled with biotin can be detected by avidin conjugated to a detectable marker. For example, avidin can be conjugated to an enzymatic marker such as alkaline phosphatase or horseradish peroxidase. In some embodiments, enzymatic markers can be detected using colorimetric rcactions using a substrate and/or a catalyst for the enzyme. In some embodiments, catalysts for alkaline phosphatase can be used, for example, 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium. In some embodiments, a catalyst can be used for horseradish peroxidase, for example, diaminobenzoate.
    • Formulations, Routes of Administration, and Effective Doses
  • Yet another aspect of the present disclosure relates to formulations, routes of administration and effective doses for pharmaceutical compositions comprising an agent or combination of agents of the instant disclosure. Such pharmaceutical compositions can be used to treat a condition (e.g., LHON) as described above.
  • Compounds of the disclosure can be administered as pharmaceutical formulations including those suitable for oral (including buccal and sub-lingual), rectal, nasal, topical, transdermal patch, pulmonary, vaginal, suppository, or parenteral (including intraocular, intravitreal, intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous and intravenous) administration or in a form suitable for administration by aerosolization, inhalation or insufflation. General information on drug delivery systems can be found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore Md. (1999).
  • In various embodiments, the pharmaceutical composition includes carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, polypeptides, amino acids, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents and/or preservatives), water, oils including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline solutions, aqueous dextrose and glycerol solutions, flavoring agents, coloring agents, detackifiers and other acceptable additives, adjuvants, or binders, other pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH buffering agents, tonicity adjusting agents, emulsifying agents, wetting agents and the like. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. In some embodiments, the pharmaceutical preparation is substantially free of preservatives. In other embodiments, the pharmaceutical preparation can contain at least one preservative. General methodology on pharmaceutical dosage forms is found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott, Williams, & Wilkins, Baltimore Md. (1999)). It can be recognized that, while any suitable carrier known to those of ordinary skill in the art can be employed to administer the compositions of this disclosure, the type of carrier can vary depending on the mode of administration.
  • Compounds can also be encapsulated within liposomes using well-known technology. Biodegradable microspheres can also be employed as carriers for the pharmaceutical compositions of this disclosure. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268, 5,075,109, 5,928,647, 5,811,128, 5,820,883, 5,853,763, 5,814,344 and 5,942,252.
  • The compound can be administered in liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to a subject are well known to those of skill in the art. U.S. Pat. No. 4,789,734, the contents of which are hereby incorporated by reference, describes methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, and along with surfactants if required, and the material dialyzed or sonicated, as necessary. A review of known methods is provided by G. Gregoriadis, Chapter 14, “Liposomes.” Drug Carriers in Biology and Medicine, pp. 2.sup.87-341 (Academic Press, 1979).
  • Microspheres formed of polymers or polypeptides are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the blood stream. Alternatively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time ranging from days to months. See, for example, U.S. Pat. Nos. 4,906,474, 4,925,673 and 3,625,214, and Jein, TIPS 19:155-157 (1998), the contents of which are hereby incorporated by reference.
  • The concentration of drug can be adjusted, the pH of the solution buffered and the isotonicity adjusted to be compatible with intraocular or intravitreal injection.
  • The compounds of the disclosure can be formulated as a sterile solution or suspension, in suitable vehicles. The pharmaceutical compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. Suitable formulations and additional carriers are described in Remington “The Science and Practice of Pharmacy” (20th Ed., Lippincott Williams & Wilkins, Baltimore Md.), the teachings of which are incorporated by reference in their entirety herein.
  • The agents or their pharmaceutically acceptable salts can be provided alone or in combination with one or more other agents or with one or more other forms. For example, a formulation can comprise one or more agents in particular proportions, depending on the relative potencies of each agent and the intended indication. For example, in compositions for targeting two different host targets, and where potencies are similar, about a 1:1 ratio of agents can be used. The two forms can be formulated together, in the same dosage unit e.g., in one cream, suppository, tablet, capsule, aerosol spray, or packet of powder to be dissolved in a beverage; or each form can be formulated in a separate unit, e.g., two creams, two suppositories, two tablets, two capsules, a tablet and a liquid for dissolving the tablet, two aerosol sprays, or a packet of powder and a liquid for dissolving the powder, etc.
  • The term “pharmaceutically acceptable salt” means those salts which retain the biological effectiveness and properties of the agents used in the present disclosure, and which are not biologically or otherwise undesirable.
  • Typical salts are those of the inorganic ions, such as, for example, sodium, potassium, calcium, magnesium ions, and the like. Such salts include salts with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid or maleic acid. In addition, if the agent(s) contain a carboxyl group or other acidic group, it can be converted into a pharmaceutically acceptable addition salt with inorganic or organic bases. Examples of suitable bases include sodium hydroxide, potassium hydroxide, ammonia, cyclohexylamine, dicyclohexyl-amine, ethanolamine, diethanolamine, triethanolamine, and the like.
  • A pharmaceutically acceptable ester or amide refers to those which retain biological effectiveness and properties of the agents used in the present disclosure, and which are not biologically or otherwise undesirable. Typical esters include ethyl, methyl, isobutyl, ethylene glycol, and the like. Typical amides include unsubstituted amides, alkyl amides, dialkyl amides, and the like.
  • In some embodiments, an agent can be administered in combination with one or more other compounds, forms, and/or agents. e.g., as described above. Pharmaceutical compositions with one or more other active agents can be formulated to comprise certain molar ratios. For example, molar ratios of about 99:1 to about 1:99 of a first active agent to the other active agent can be used. In some subset of the embodiments, the range of molar ratios of a first active agent: other active agents are selected from about 80:20 to about 20:80; about 75:25 to about 25:75, about 70:30 to about 30:70, about 66:33 to about 33:66, about 60:40 to about 40:60; about 50:50; and about 90:10 to about 10:90. The molar ratio of a first active: other active agents can be about 1:9, and in some embodiments can be about 1:1. The two agents, forms and/or compounds can be formulated together, in the same dosage unit e.g., in one cream, suppository, tablet, capsule, or packet of powder to be dissolved in a beverage; or each agent, form, and/or compound can be formulated in separate units, e.g., two creams, suppositories, tablets, two capsules, a tablet and a liquid for dissolving the tablet, an aerosol spray a packet of powder and a liquid for dissolving the powder, etc.
  • If necessary or desirable, the agents and/or combinations of agents can be administered with still other agents. The choice of agents that can be co-administered with the agents and/or combinations of agents of the instant disclosure can depend, at least in part, on the condition being treated.
  • The agent(s) (or pharmaceutically acceptable salts, esters or amides thereof) can be administered per se or in the form of a pharmaceutical composition wherein the active agent(s) is in an admixture or mixture with one or more pharmaceutically acceptable carriers. A pharmaceutical composition, as used herein, can be any composition prepared for administration to a subject. Pharmaceutical compositions for use in accordance with the present disclosure can be formulated in conventional manner using one or more physiologically acceptable carriers, comprising excipients, diluents, and/or auxiliaries, e.g., which facilitate processing of the active agents into preparations that can be administered. Proper formulation can depend at least in part upon the route of administration chosen. The agent(s) useful in the present disclosure, or pharmaceutically acceptable salts, esters, or amides thereof, can be delivered to a subject using a number of routes or modes of administration, including oral, buccal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous, intraocular, intravitreal, and intramuscular applications, as well as by inhalation.
  • In some embodiments, oils or non-aqueous solvents can be used to bring the agents into solution, due to, for example, the presence of large lipophilic moieties. Alternatively, emulsions, suspensions, or other preparations, for example, liposomal preparations, can be used. With respect to liposomal preparations, any known methods for preparing liposomes for treatment of a condition can be used. See, for example, Bangham et al., J. Mol. Biol. 23: 238-252 (1965) and Szoka et al., Proc. Natl Acad. Sci. USA 75: 4194-4198 (1978), incorporated herein by reference. Ligands can also be attached to the liposomes to direct these compositions to particular sites of action. Agents of this disclosure can also be integrated into foodstuffs, e.g., cream cheese, butter, salad dressing, or ice cream to facilitate solubilization, administration, and/or compliance in certain subject populations.
  • The compounds of the disclosure can be formulated for parenteral administration (e.g., by injection, for example, intraocular or intravitreal injection) and can be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example, solutions in aqueous polyethylene glycol.
  • For injectable formulations, the vehicle can be chosen from those known in art to be suitable, including aqueous solutions or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. The formulation can also comprise polymer compositions which are biocompatible, biodegradable, such as poly(lactic-co-glycolic)acid. These materials can be made into micro or nanospheres, loaded with drug and further coated or derivatized to provide superior sustained release performance. Vehicles suitable for periocular or intraocular injection include, for example, suspensions of therapeutic agent in injection grade water, liposomes and vehicles suitable for lipophilic substances. Other vehicles for periocular or intraocular injection are well known in the art.
  • In some embodiments, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
  • When administration is by injection, the active compound can be formulated in aqueous solutions, specifically in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer. The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active compound can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In some embodiments, the pharmaceutical composition does not comprise an adjuvant or any other substance added to enhance the immune response stimulated by the peptide. In some embodiments, the pharmaceutical composition comprises a substance that inhibits an immune response to the peptide. Methods of formulation are known in the art, for example, as disclosed in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton P.
  • In some embodiments, eye disorders can be effectively treated with ophthalmic solutions, suspensions, ointments or inserts comprising an agent or combination of agents of the present disclosure. Eye drops can be prepared by dissolving the active ingredient in a sterile aqueous solution such as physiological saline, buffering solution, etc., or by combining powder compositions to be dissolved before use. Other vehicles can be chosen, as is known in the art, including but not limited to: balance salt solution, saline solution, water soluble polyethers such as polyethyene glycol, polyvinyls, such as polyvinyl alcohol and povidone, cellulose derivatives such as methylcellulose and hydroxypropyl methylcellulose, petroleum derivatives such as mineral oil and white petrolatum, animal fats such as lanolin, polymers of acrylic acid such as carboxypolymethylene gel, vegetable fats such as peanut oil and polysaccharides such as dextrans, and glycosaminoglycans such as sodium hyaluronate. If desired, additives ordinarily used in the eye drops can be added. Such additives include isotonizing agents (e.g., sodium chloride, etc.), buffer agent (e.g., boric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc.), preservatives (e.g., benzalkonium chloride, benzethonium chloride, chlorobutanol, etc.), thickeners (e.g., saccharide such as lactose, mannitol, maltose, etc.; e.g., hyaluronic acid or its salt such as sodium hyaluronate, potassium hyaluronate, etc.; e.g., mucopolysaccharide such as chondroitin sulfate, etc.; e.g., sodium polyacrylate, carboxyvinyl polymer, crosslinked polyacrylate, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propyl cellulose or other agents known to those skilled in the art).
  • The solubility of the components of the present compositions can be enhanced by a surfactant or other appropriate co-solvent in the composition. Such cosolvents include polysorbate 20, 60, and 80, Pluronic F68, F-84 and P-103, cyclodextrin, or other agents known to those skilled in the art. Such co-solvents can be employed at a level of from about 0.01% to 2% by weight.
  • The compositions of the disclosure can be packaged in multidose form. Preservatives can be preferred to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, Onamer M. or other agents known to those skilled in the art. In the prior art ophthalmic products, such preservatives can be employed at a level of from 0.004% to 0.02%. In the compositions of the present application the preservative, preferably benzalkonium chloride, can be employed at a level of from 0.001% to less than 0.01%, e.g., from 0.001% to 0.008%, preferably about 0.005% by weight. It has been found that a concentration of benzalkonium chloride of 0.005% can be sufficient to preserve the compositions of the present disclosure from microbial attack.
  • In some embodiments, the agents of the present disclosure are delivered in soluble rather than suspension form, which allows for more rapid and quantitative absorption to the sites of action. In general, formulations such as jellies, creams, lotions, suppositories and ointments can provide an area with more extended exposure to the agents of the present disclosure, while formulations in solution, e.g., sprays, provide more immediate, short-term exposure.
  • It is envisioned additionally, that the compounds of the disclosure can be attached releasably to biocompatible polymers for use in sustained release formulations on, in or attached to inserts for topical, intraocular, periocular, or systemic administration. The controlled release from a biocompatible polymer can be utilized with a water soluble polymer to form an instillable formulation, as well. The controlled release from a biocompatible polymer, such as for example, PLGA microspheres or nanospheres, can be utilized in a formulation suitable for intra ocular implantation or injection for sustained release administration, as well any suitable biodegradable and biocompatible polymer can be used.
    • EXAMPLES
  • The following exemplary embodiments further describe the present invention. It should be understood that these examples are only intended to illustrate the invention, but not to limit the scope of the present invention. Unless otherwise indicated, the methods and conditions disclosed in e.g., sambrook et al, molecular cloning: a laboratory manual (New York: cold spring harbor laboratory press, 1989) or the conditions recommended by the manufacturer can be used in the examples below.
    • Example 1—ND4 Plasmid and Virus Preparation
  • 1.1 Plasmid Preparation
  • The nucleotide sequence for human ND4 (SEQ ID NO: 6) was obtained based on US National Center for Biotechnology Information reference sequence yp_003024035.1. The sequences for the non-optimized mitochondrial targeting sequence COX10 is SEQ ID NO: 1. The optimized sequences for the mitochondrial targeting sequence COX10 (opt_COX10, SEQ ID NO: 2) and the coding sequence of human ND4 (opt_ND4, SEQ ID NO: 7) were designed to improve the transcription efficiency and the translation efficiency. The optimized COX10-ND4 sequence, which is about 75.89% homology to the non-optimized COX10-ND4, was followed by a three prime untranslated region (i.e., 3′UTR, SEQ ID NO: 13) to a recombinant nucleic acid, opt_COX10-opt_ND4-3′UTR (as shown in SEQ ID NO: 31).
  • The synthesized recombinant nucleic acid, opt_COX10-opt_ND4-3′UTR, was incorporated into an adeno-associated virus (AAV) vector by PCR amplification (FIG. 1). The opt_COX10-opt_ND4-3′UTR was cut by the EcoRI/SalI restriction enzymes to form cohesive ends, and then embedded into an AAV vector with EcoRI/SalI restriction sites, such as the pSNaV vector, to generate the pSNaV/rAAV2/2-ND4 plasmid (i.e., the pAAV2-optimized ND4 plasmid). The pAAV2-opt_ND4 plasmid was compared to the non-optimized pAAV2-ND4 plasmid.
  • The recon screening and identifying steps were similar to the CN102634527B: the plasmid was cultured at 37° C. in a LB plate. Blue colonies and white colonies were appeared, where white colonies were recombinant clones. The white colonies were picked, added to 100 mg/L ampicillin-containing LB culture medium, cultured at 37° C., 200 rpm for 8 hours and then the plasmid were extracted from the cultured bacterial medium based on the Biomiga plasmid extraction protocol. The identification of the plasmid was confirmed using the EcoRI/SalI restriction enzymes.
  • 1.2 Cell Transfection
  • One day before transfection, HEK293 cells were inoculated to 225 cm2 cell culture bottle: at the inoculation density of 3.0×107 cells/ml, the culture medium was the Dulbecco's Modified Eagle Medium (DMEM) with 10% bovine serum, at 37° C. in a 5% CO2 incubator overnight. The culture medium were replaced with fresh DMEM with 10% bovine serum on the day of transfection.
  • After the cells grow to 80-90%, discard the culture medium and transfect the cells with the pAAV2-ND4 and pAAV2-opt_ND4 plasmid, using the PlasmidTrans (VGTC) transfection kit. The detailed transfection protocol was described in CN102634527B example 1. The cells were collected 48 h after the transfection.
  • 1.3 Collection, Concentration and Purification of the Recombinant Adeno-Associated Virus
  • Virus collection: 1) dry ice ethanol bath (or liquid nitrogen) and a 37° C. water bath were prepared; 2) the transfected cells along with media were collected in a 15 ml centrifuge tube, 3) the cells were centrifuged for 3 minutes at 1000 rpm/min; the cells and supernatant were separated, the supernatant were stored separately; and the cells were re-suspended in 1 ml of PBS: 4) the cell suspension were transferred between the dry ice-ethanol bath and 37° C. water bath repeatedly, freeze thawing for four times for 10 minutes each, slightly shaking after each thawing.
  • Virus concentration: 1) cell debris were removed with 10,000 g centrifugation; the centrifugal supernatant was transferred to a new centrifuge tube; 2) impurities were removed by filtering with a 0.45 μm filter; 3) each ½ volume of 1M NaCl and 10% PEG 8000 solution were added in the sample, uniformly mixed, and stored at 4° C. overnight; 4) supernatant was discarded after 12,000 rpm centrifugation for 2 h; after the virus precipitate was completely dissolving in an appropriate amount of PBS solution, sterilizing the sample with a 0.22 μm filter; 5) adding benzonase nuclease was added to remove residual plasmid DNA (final concentration at 50 U/ml). The tube was inverted several times to mix thoroughly and then incubated at 37° C. for 30 minutes; 6) the sample was filtered with a 0.45 μm filtration head; the filtrate is the concentrated rAAV2 virus.
  • Virus purification: 1) CsCl was added to the concentrated virus solution until a density of 1.41 g/ml (refraction index at 1.372): 2) the sample was added to in the ultracentrifuge tube and filled the tube with pre-prepared 1.41 g/ml CsCl solution: 3) centrifuged at 175,000 g for 24 hours to form a density gradient. Sequential collection of different densities of the sample was performed. The enriched rAAV2 particles were collected; 4) repeating the process one more time. The virus was loaded to a 100 kDa dialysis bag and dialyzed/desalted at 4° C. overnight. The concentrated and purified recombinant adeno-associated virus were rAAV2-ND4 and rAAV2-optimized ND4.
  • Similarly, other mitochondrial targeting sequences (MTS), such as OPA1 (SEQ ID NO: 5) can be used to replace COX10 in the above example and create AAV with recombinant plasmids.
    • Example 2—Intravitreal Injection of rAAV2 in Rabbit Eyes
  • Twelve rabbits were divided into 2 group: rAAV2-ND4 and rAAV2-optimized ND4. Virus solution (1-101 vg/0.05 mL) was punctured into the vitreous cavity from 3 mm outside the corneal limbus at the pars plana. After the intravitreal injection, the eyes were examined using slit lamp exam and fundus photography inspection. Injection for 30 days. RT-PCR detection and immunoblotting were carried out in each group respectively.
    • Example 3—Real-Time PCR for the Expression of ND4
  • The RNAs from the transfected rAAV2-ND4 and rAAV2-optimized ND4 rabbit optic nerve cells were extracted using the TRIZOL total RNA extraction kit. cDNA templates were synthesized by reverse transcription of the extracted RNA.
  • The NCBI conserved structural domain analysis software were used to analyze the conservative structure of ND4, ensuring that the designed primers amplified fragments were located at non-conserved region; then primers were designed according to the fluorescent quantitative PCR primer design principle:
  • β-actin-S:
    (SEQ ID NO: 85)
    CGAGATCGTGCGGGACAT;
    β-actin-A:
    (SEQ ID NO: 86)
    CAGGAAGGAGGGCTGGAAC;
    ND4-S:
    (SEQ ID NO: 87)
    CTGCCTACGACAAACAGAC;
    ND4-A:
    (SEQ ID NO: 88)
    AGTGCGTTCGTAGTTTGAG;
  • The fluorescent quantitative PCR reaction and protocol: fluorescence quantitative PCR were measured in a real-time PCR detection system. In a 0.2 ml PCR reaction tube, SYBR green mix 12.5 W, ddH2O 8 μl, 1 sW of each primer, and the cDNA sample 2.5 μl, were added to an overall volume of 25 μl. Each sample was used for amplification of the target gene and amplifying the reference gene β-actin, and each amplification were repeated three times. The common reagents were added together and then divided separately to minimize handling variation. The fluorescent quantitative PCR were carried out: pre-denaturation at 95° C. for 1 s, denaturation at 94° C. for 15 s, annealing at 55° C. for 15 sec, extension at 72° C. for 45 s. A total of 40 cycles of amplification reaction were performed and fluorescence signal acquisition was done at the extension phase of each cycle. After the reaction, a 94° C. to 55° C. melting curve analysis was done. By adopting a relative quantitative method research of gene expression level difference to beta-actin was used as an internal reference gene.
  • As shown in FIG. 2, the relative expression level (mRNA level) of the rAAV2-ND4 and rAAV2-optimized ND4 were 0.42±0.23 and 0.57±0.62, respectively (p<0.05, FIG. 2). The results unexpectedly show that the optimized ND4 (opt_ND4, SEQ ID NO: 7) coding nucleic acid sequence and the corresponding recombinant nucleic acid (opt_COX10-opt_ND4-3′UTR, SEQ ID NO: 31) surprisingly increased the transcription efficiency, increasing the expression of the rAAV2-optimized ND4 by about 36%. The results showed that the transcription efficiency of the rAAV2-optimized ND4 is significantly higher.
    • Example 4—Immunoblotting Detection of ND4 Expression
  • The ND4 protein was purified from the rabbit nerve cells transfected by rAAV2-optimized ND4 and rAAV2-ND4, respectively. After a 10% polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane (Bio-Rad, HER-hercules, CA, USA) for immune detection. β-actin was used as an internal reference gene. The film strip was observed on an automatic image analysis instrument (Li-Cor; Lincoln, Nebr., USA) and analyzed using the integrated optical density of the protein band with integral normalization method, so as to obtain the same sample corresponding optical density value. The statistical analysis software SPSS 19.0 was used for the data analysis.
  • The results was shown in FIG. 3. The average relative protein expression level of ND4 for rAAV2-optimized ND4 (left black column) and rAAV2-ND4 was 0.32±0.11 and 0.68 t 0.20, respectively (p<0.01, FIG. 3). The results unexpectedly show that the optimized ND4 coding nucleic acid sequence (opt_ND4, SEQ ID NO: 7) and the corresponding recombinant nucleic acid (opt_COX10-opt_ND4-3′UTR, SEQ ID NO: 31) surprisingly increased the translation efficiency, increasing the expression of the rAAV2-optimized ND4 by about 112%. The results showed that the translation efficiency of the rAAV2-optimized ND4 is also significantly higher.
    • Example 5—Rabbits Intraocular Pressure and Eye-Ground Photography
  • Slit lamp examination and intraocular pressure measurement was performed on both groups of rabbits at 1, 3, 7, and 30 days after the surgery. No obviously abnormality, conjunctival congestion, secretions, or endophthalmitis were observed and the intraocular pressure were not elevated in all the rabbits.
  • The fundus photographic results were shown in FIG. 4. No obvious damage or complication to the optic nerve and retinal vascular of the rabbits, indicating the standard intravitreal injection is safe without noticeable inflammation reaction or other complications.
    • Example 6—Human Clinical Trial
  • Two groups of patients were tested: 1) between 2011 and 2012, 9 patients received intravitreal injection of 1-1010 vg/0.05 mL rAAV2-ND4 in a single eye, as a control group; and 2) between 2017 and January 2018, 20 patients received intravitreal injection of 1×1010 vg/0.05 mL rAAV2-optimized ND4 in a single eye, as an experimental group. The results of the clinical trial were analyzed using the statistical analysis SPSS 19.0.
  • The comparison of the two groups is shown in Table 2. The fastest eyesight improving time was 1 month in the experimental group, which was significantly faster than the control group at 3 months (p<0.01): the optimal recovery of vision for the experimental group was 1.0, which was obviously higher than the control group at 0.8 (p<0.01); the average recovery of vision in the experimental group was 0.582±0.086, which was obviously higher than the control group at 0.344±0.062 (p<0.01). The fundus photographic results were shown in FIG. 5. No obvious damage or complication to the optic nerve and retinal vascular of the patients in the experimental and control groups, indicating the safety of the intravitreal injection of rAAV2-optimized ND4 and rAAV2-ND4.
  • TABLE 2
    The comparison of rAAV2-optimized ND4 and rAAV2-ND4 in LHON gene therapy
    Fastest eyesight Number of optimal average
    Patient improving time patients with recovery of recovery of
    group number (month) improved vision vision vision
    control 9 3  6 (67%) 0.8 0.344 ± 0.062
    experimental 20 1 15 (75%) 1.0 0.582 ± 0.086
    P value <0.01 <0.01 <0.01 <0.01
    • Example 7—OPA1 as the Mitochondrial Targeting Sequences
  • The COX10 and 3′UTR sequences in the recombinant nucleic acid (opt_COX10-opt_ND4-3′UTR, SEQ ID NO: 31) in examples 1-6 were replaced with another mitochondrial targeted sequence, OPA1 (SEQ ID NO: 5) and another 3′UTR sequence, 3′UTR* (SEQ ID NO: 14) respectively, to generate a new recombinant nucleic acid, OPA1-opt_ND4-3′UTR* (SEQ ID NO: 74).
  • Experimental methods were the same as examples 1-6, where the recombinant nucleic acid opt_COX10-opt_ND4-3′UTR (SEQ ID NO: 31) was replaced by OPA1-opt_ND4-3′UTR* (SEQ ID NO: 74). It was found that, the optimized ND4 sequence has significantly improved transcription and translation efficiencies, expression levels, as well as higher efficacy and safety in treating LHON when compared to non-optimized ND4 (COX10-ND4-3′UTR, SEQ ID NO: 15).
    • Example 8—Optimized ND4 Sequence Opt_ND4*
  • Similar experimental methods in examples 1-6 were followed using the nucleic acid, opt_COX10*-opt_ND4*-3′UTR (SEQ ID NO: 47). Follow the similar procedures as in example 1, virus tagged with a fluorescent protein. EGFP, was prepared as rAAV2-ND4-EGFP and rAAV2-opt_ND4*-EGFP.
  • The frozen 293T cell was resuscitated and allowed to grow in a T75 flask to about 90%. The cells were precipitated and resuspended in DMEM complete medium to a cell density of 5×104 cells/mL. The cells were resuspended. About 100 μl of the cell suspension (about 5000 cells) were added in each well of a 96 well plate. The cells were cultured and grown to 50% under 37° C. and 5% CO2. About 0.02 μl PBS was mixed with 2×10 μl vg/0.02 μl of the virus rAAV2-ND4-EGFP and rAAV2-opt_ND4*-EGFP, respectively. After 48 hours, fluorescence microscopy and RT-PCR detection and immunoblotting experiments were performed. As shown in FIG. 6, EGFP was successfully expressed, indicating that rAAV carrying the EGFP gene was successfully transfected in the 293T cells and rAAV2-ND4-EGFP and rAAV2-opt_ND4*-EGFP were successfully expressed.
  • Real-time PCR tests similar to example 3 was following using the following primers:
  • β-actin-S:
    (SEQ ID NO: 85)
    CGAGATCGTGCGGGACAT;
    β-actin-A:
    (SEQ ID NO: 86)
    CAGGAAGGAGGGCTGGAAC;
    ND4-S:
    (SEQ ID NO: 107)
    GCCAACAGCAACTACGAGC;
    ND4-A:
    (SEQ ID NO: 108)
    TGATGTTGCTCCAGCTGAAG;
  • The results unexpectedly show that the optimized ND4* (opt_ND4, SEQ ID NO: 8) coding nucleic acid sequence and the corresponding recombinant nucleic acid (opt_COX10*-opt ND4*-3′UTR, SEQ ID NO: 47) surprisingly increased the transcription efficiency, increasing the expression of the rAAV2-opt_ND4 by about 20%. The results showed that the transcription efficiency of the rAAV2-opt_ND4 is significantly higher.
  • FIG. 7 shows the ND4 expression in 293T cells. The average expression of ND4 protein for rAAV2-ND4 is 0.36, while the average expression of ND4 protein for rAAV2-opt_ND4* is 1.65, which is about 4.6 times higher than the rAAV2-ND4 group (p<0.01) (see FIG. 8).
  • FIG. 9 shows the ND4 expression in rabbit optic nerve cells. The average expression of ND4 protein for rAAV2-ND4 is 0.16, while the average expression of ND4 protein for rAAV2-opt_ND4* is 0.48, which is about 3 times higher than the rAAV2-ND4 group (p<0.01) (see FIG. 10).
  • Similar to example 5, slit lamp examination and intraocular pressure measurement was performed on both groups of rabbits at 1, 3, 7, and 30 days after the surgery. No obviously abnormality, conjunctival congestion, secretions, or endophthalmitis were observed and the intraocular pressure were not elevated in all the rabbits.
  • The fundus photographic results for rAAV2-ND4 and rAAV2-opt_ND4* were shown in FIG. 11. No obvious damage or complication to the optic nerve and retinal vascular of the rabbits, indicating the standard intravitreal injection is safe without noticeable inflammation reaction or other complications.
  • Eye balls from both rabbit groups were removed after the slit lamp examination and intraocular pressure measurement. Eye balls were fixed, and dehydrated using paraffin. Tissues were pathologically sectioned along the direction of optic nerves. After further dehydration, the tissue sample was dyed using hematoxylin and eosin. The microscope inspection result is referred to FIG. 12. As shown in the HE staining results, the rabbit retinal ganglion fiber layer was not damaged and the number of ganglion cells was not reduced, indicating the intravitreal injection did not produce retinal toxicity or nerve damage, and can be used safely.
  • Experimental methods were the same as example 8, where the recombinant nucleic acid opt_COX10*-opt_ND4*-3′UTR (SEQ ID NO: 47) was replaced by OPA1-opt_ND4*-3′UTR* (SEQ ID NO: 76). It was found that, the optimized ND4 sequence has significantly improved transcription and translation efficiencies, expression levels, as well as higher efficacy and safety in treating LHON when compared to non-optimized ND4 (COX10-ND4-3′UTR, SEQ ID NO: 15).
    • Example 9—ND6 Sequence
  • Similar experimental methods in examples 1-6 were followed using the nucleic acid, COX10-ND6-3′UTR (SEQ ID NO: 21), which is the combination (5′ to 3′) of COX10 (SEQ ID NO: 1), ND6 (SEQ ID NO: 9), and 3′UTR (SEQ ID NO: 13).
  • The plasmid screening for COX10-ND6-3′UTR (SEQ ID NO: 21) used the following primers:
  • ND6-F:
    (SEQ ID NO: 89)
    ATGATGTATGCTTTGTTTCTG,
    ND6-R:
    (SEQ ID NO: 90)
    CTAATTCCCCCGAGCAATCTC,
  • The transfected and screened virus rAAV2-ND6 had a viral titer of 2.0×1011 vg/mL. Similar to example 5, slit lamp examination and intraocular pressure measurement was performed on three groups of rabbits (A: rAAV2-ND6; B: rAAV-GFP; C: PBS) at 1, 7, and 30 days after the surgery (FIG. 13). No obviously abnormality, conjunctival congestion, secretions, or endophthalmitis were observed and the intraocular pressure were not elevated in all the rabbits.
  • Real-time PCR tests similar to example 3 was following using the following primers:
  • β-actin-S:
    (SEQ ID NO: 85)
    CGAGATCGTGCGGGACAT;
    β-actin-A:
    (SEQ ID NO: 86)
    CAGGAAGGAGGGCTGGAAC;
    ND6-S:
    (SEQ ID NO: 91)
    AGTGTGGGTTTAGTAATG;
    ND4-A:
    (SEQ ID NO: 92)
    TGCCTCAGGATACTCCTC;
  • The results show that the expression of ND6 for rAAV2-ND6 and control (PBS) was 0.59±0.06 and 0.41±0.03, respectively. The results showed that the transcription efficiency of the rAAV2-ND6 is higher than the control group (p<0.01).
    • Example 10—Optimized Opt_ND6 Sequence
  • Similar experimental methods in examples 1-6 were followed using the nucleic acid, opt_COX10*-opt_ND6-3′UTR (SEQ ID NO: 51), which is the combination (5′ to 3′) of opt_COX10* (SEQ ID NO: 3), opt_ND6 (SEQ ID NO: 10), and 3′UTR (SEQ ID NO: 13).
  • Three groups of rabbits were injected: A: 1010 vg/50 μl of rAAV2-opt_ND6, B: 1010 vg/50 μl of rAAV2-ND6 (example 9), and C: 1010 vg/50 μl of rAAV2-EGFP. FIG. 14 shows the fundus photographic results for rabbits injected with rAAV2-opt_ND6 (A), rAAV2-ND6 (B), rAAV-EGFP (C), respectively. No obviously abnormality, conjunctival congestion, secretions, or endophthalmitis were observed and the intraocular pressure were not elevated in all the rabbits.
  • Real-time PCR tests similar to example 3 was following using the following primers:
  • β-actin-F:
    (SEQ ID NO: 93)
    CTCCATCCTGGCCTCGCTGT;
    β-actin-R:
    (SEQ ID NO: 94)
    GCTGTCACCTTCACCGTTCC;
    ND6-F:
    (SEQ ID NO: 95)
    GGGTTTTCTTCTAAGCCTTCTCC;
    ND6-R:
    (SEQ ID NO: 96)
    CCATCATACTCTTTCACCCACAG;
    opt_ND6-F:
    (SEQ ID NO: 97)
    CGCCTGCTGACCGGCTGCGT;
    opt_ND6-R:
    (SEQ ID NO: 98)
    CCAGGCCTCGGGGTACTCCT;
  • As shown in FIG. 15, rAAV2-opt_ND6 (A) and rAAV2-ND6 (B) both had higher (p<0.05) relative ND6 expression levels than the control group (C). rAAV2-opt_ND6 (A) had a little higher relative ND6 expression levels than rAAV2-ND6 (B). As shown in the western blot in FIG. 16, rAAV2-opt_ND6 (A) had more than 3 times higher relative ND6 expression levels than rAAV2-ND6 (B).
  • Experimental methods were the same as example 8, where the recombinant nucleic acids, COX10-ND6-3′UTR (SEQ ID NO: 21) and opt_COX10*-opt_ND6-3′UTR (SEQ ID NO: 51), were replaced by OPA1-ND6-3′UTR (SEQ ID NO: 77) and OPA1-opt_ND6-3′UTR (SEQ ID NO: 79). It was found that, the optimized ND6 sequence has significantly improved transcription and translation efficiencies, expression levels, as well as higher efficacy and safety in treating LHON.
    • Example 11—ND1 and opt_ND1 Sequences
  • Similar experimental methods in examples 1-6 were followed using rAAV2-ND1, COX10-ND1-3′UTR (SEQ ID NO: 25), which is the combination (5′ to 3′) of COX10 (SEQ ID NO: 1), ND1 (SEQ ID NO: 11), and 3′UTR(SEQ ID NO: 13); and rAAV2-opt_ND1, opt_COX10*-opt_ND1-3′UTR (SEQ ID NO: 55), which is the combination (5′ to 3′) of opt_COX10* (SEQ ID NO: 3), opt_ND1 (SEQ ID NO: 12), and 3′UTR (SEQ ID NO: 13).
  • The plasmid screening for COX10-ND1-3′UTR (SEQ ID NO: 25) used the following primers:
  • ND1-F:
    (SEQ ID NO: 99)
    ATGGCCGCATCTCCGCACACT,
    ND1-R:
    (SEQ ID NO: 100)
    TTAGGTTTGAGGGGGAATGCT,
  • The plasmid screening for opt_COX10*-opt_ND1-3′UTR (SEQ ID NO: 55) used the following primers:
  • ND1-F:
    (SEQ ID NO: 101)
    AACCTCAACCTAGGCCTCCTA,
    ND1-R:
    (SEQ ID NO: 102)
    TGGCAGGAGTAACCAGAGGTG,
  • Three groups of rabbits were injected: A: 10′0 vg/50 μl of rAAV2-opt_ND1, B: 1010 vg/50 μl of rAAV2-ND1 (example 9), and C: 1010 vg/50 μl of rAAV2-EGFP. No obviously abnormality, conjunctival congestion, secretions, or endophthalmitis were observed and the intraocular pressure were not elevated in all the rabbits.
  • Real-time PCR tests similar to example 3 was following using the following primers:
  • ND1-F:
    (SEQ ID NO: 103)
    AGGAGGCTCTGTCTGGTATCTTG;
    ND1-R:
    (SEQ ID NO: 104)
    TTTTAGGGGCTTCTTTGGTGAA;
    opt_ND1-F:
    (SEQ ID NO: 105)
    GCCGCCTGCTGACCGGCTGCGT;
    opt_ND1-R:
    (SEQ ID NO: 106)
    TGATGTACAGGGTGATGGTGCTGG;
  • As shown in FIG. 17, rAAV2-opt_ND1 (A) and rAAV2-ND1 (B) both had higher (p<0.05) relative ND1 expression levels than the control group (C). As shown in the western blot in FIG. 18, rAAV2-opt_ND1 (A) had more than 2 times higher relative ND6 expression levels than rAAV2-ND1 (B).
  • Experimental methods were the same as example 8, where the recombinant nucleic acids, COX10-ND1-3′UTR (SEQ ID NO: 25) and opt_COX10*-opt_ND1-3′UTR (SEQ ID NO: 55), were replaced by OPA1-ND1-3′UTR (SEQ ID NO: 81) and OPA1-opt_ND1-3′UTR (SEQ ID NO: 83). It was found that, the optimized ND1 sequence has significantly improved transcription and translation efficiencies, expression levels, as well as higher efficacy and safety in treating LHON.
    • Example 12—Other Fusion Proteins
  • Similar experimental methods in examples 1-6 can be followed using other fusion proteins as set forth in SEQ ID NO: 15-84. And similar results are expected to be achieved.
    • Example 13—Formulation Development
  • AAV2 virus samples were used to screen different AAV formulations. The stability of the different AAV formulations were evaluated using the StepOnePlus real-time PCR system. The viral titer of each formulation under a freeze/thaw cycle condition was measured.
  • First, three different formulations were tested under 1, 2, 3, 4, and 5 freeze/thaw cycles and the viral titers were measured and summarized in Table 3. The three formulations tested were: A: phosphate-buffered saline (PBS); B: 1% α,α-trehalose dehydrate, 1% L-histidine monohydrochloride monohydrate, and 1% polysorbate 20; and C: 180 mM NaCl, 10 mM NaH2PO4/Na2HPO4, and 0.001% poloxamer 188, pH 7.3. As shown in Table 3, formulation C has the lowest relative standard deviation (RSD) after 5 freeze/thaw cycles, indicating superior stability as an AAV formulation.
  • TABLE 3
    the viral titers of formulations A, B, and C
    viral titers 0 cycle 1 cycle 2 cycles 3 cycles 4 cycles 5 cycles RSD
    A 1.15E+11 9.48E+10 6.16E+10 2.90E+10 1.56E+10 5.26E+09 83.18
    B 4.25E+11 5.12E+11 6.66E+11 4.30E+11 4.77E+11 4.20E+11 19.30
    C 4.96E+11 6.91E+11 7.69E+11 6.82E+11 6.83E+11 7.27E+11 13.90
  • As shown in Table 3, formulation C has the lowest relative standard deviation (RSD) after 5 freeze/thaw cycles, indicating superior stability as an AAV formulation.
  • Second, another group of three different formulations were tested under 1, 2, 3, 4, and 5 freeze/thaw cycles and the viral titers were measured and summarized in Table 4. The three formulations tested were: D: phosphate-buffered saline (PBS), pH 7.2-7.4; E: PBS and 0.001% poloxamer 188, pH 7.2-7.4; and F: 80 mM NaCl, 5 mM NaH2PO4, 40 mM Na2HPO4, 5 mM KH2PO4 and 0.001% poloxamer 188, 7.2-7.4.
  • TABLE 4
    the viral titers of formulations D, E, and F
    viral titers 0 cycle 1 cycle 2 cycles 3 cycles 4 cycles 5 cycles RSD
    D 1.13E+10 4.62E+09 2.25E+09 1.25E+09 1.01E+09 9.48E+08 113.25
    E 4.72E+10 5.48E+10 5.33E+10 5.33E+10 4.94E+10 4.08E+10 10.53
    F 6.61E+10 6.08E+10 6.47E+10 6.84E+10 6.52E+10 6.05E+10 4.81
  • As shown in Table 4, formulation F has the lowest relative standard deviation (RSD) after 5 freeze/thaw cycles, indicating superior stability as an AAV formulation. Overall, formulation F also has the lowest RSD among all tested formulations and can be used as the AAV formulation for future development.
  • While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims (29)

1-139. (canceled)
140. A pharmaceutical composition, comprising:
an adeno-associated virus (AAV) comprising a recombinant nucleic acid comprising a sequence that is at least 90% identical to a sequence as set forth in SEQ ID NO: 15; and
a pharmaceutically acceptable excipient comprising phosphate-buffered saline (PBS), α,α-trehalose dehydrate, L-histidine monohydrochloride monohydrate, polysorbate 20, poloxamer 188, or any combination thereof.
141. The pharmaceutical composition of claim 140, wherein said pharmaceutically acceptable excipient comprises poloxamer 188.
142. The pharmaceutical composition of claim 141, wherein said pharmaceutically acceptable excipient comprises 0.0001%-0.01% poloxamer 188.
143. The pharmaceutical composition of claim 142, wherein said pharmaceutically acceptable excipient comprises 0.001% poloxamer 188.
144. The pharmaceutical composition of claim 140, wherein said pharmaceutically acceptable excipient further comprises one or more salts.
145. The pharmaceutical composition of claim 144, wherein said one or more salts comprises NaCl, NaH2PO4, Na2HPO4, or KH2PO4.
146. The pharmaceutical composition of claim 145, wherein said one or more salts comprises NaCl, NaH2PO4, Na2HPO4, and KH2PO4.
147. The pharmaceutical composition of claim 146, wherein said one or more salts comprises 80 mM NaCl, 5 mM NaH2PO4, 40 mM Na2HPO4, and 5 mM KH2PO4.
148. The pharmaceutical composition of claim 140, wherein said pharmaceutical composition has a pH of 6-8.
149. The pharmaceutical composition of claim 148, wherein said pharmaceutical composition has a pH of 7.2-7.4.
150. The pharmaceutical composition of claim 149, wherein said pharmaceutical composition has a pH of 7.3.
151. The pharmaceutical composition of claim 140, wherein said pharmaceutical composition has a viral titer of at least 1.0×1010 vg/mL.
152. The pharmaceutical composition of claim 151, wherein said pharmaceutical composition has a viral titer of at least 5.0×1010 vg/mL.
153. The pharmaceutical composition of claim 140, when said pharmaceutical composition is subject to five freeze/thaw cycles, said pharmaceutical composition retains at least 60% of a viral titer as compared to the viral titer prior to the five freeze/thaw cycles.
154. The pharmaceutical composition of claim 140, wherein said pharmaceutical composition, when administered to a patient with Leber's hereditary optic neuropathy, generates a higher average recovery of vision than a comparable pharmaceutical composition without said recombinant nucleic acid.
155. A method of treating Leber's hereditary optic neuropathy (LHON), comprising administering the pharmaceutical composition of claim 140 to a patient in need thereof.
156. The method of claim 155, wherein said pharmaceutical composition is administered via intravitreal injection.
157. The method of claim 156, wherein about 0.01-0.1 mL of said pharmaceutical composition is administered via intravitreal injection.
158. The method of claim 157, wherein about 0.05 mL of said pharmaceutical composition is administered via intravitreal injection.
159. The method of claim 155, further comprising administering methylprednisolone to said patient.
160. The method of claim 159, wherein said methylprednisolone is administered intravenously or orally.
161. The method of claim 160, comprising administering methylprednisolone intravenously for at least one day, which is followed by administering methylprednisolone orally for at least a week.
162. The method of claim 161, comprising administering methylprednisolone intravenously for about 3 days, which is followed by administering methylprednisolone orally for at least about 6 weeks.
163. The method of claim 159, wherein said methylprednisolone is administered daily for at least 2 days prior to said intravitreal injection of said pharmaceutical composition.
164. The method of claim 159, wherein said methylprednisolone is administered intravenously at a daily dose of about 80 mg/60 kg.
165. The method of claim 155, further comprising administering creatine phosphate sodium to said patient.
166. The method of claim 165, wherein said creatine phosphate sodium is administered intravenously.
167. The method of claim 155, wherein said administering said pharmaceutical composition generates a higher average recovery of vision than a comparable pharmaceutical composition without said recombinant nucleic acid.
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